LOCUS       40924_32340        7889 bp DNA     circular SYN 13-MAY-2021
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7889)
  AUTHORS   Koo BK, Stange DE, Sato T, Karthaus W, Farin HF, Huch M, van Es JH, 
            Clevers H
  TITLE     Controlled gene expression in primary Lgr5 organoid cultures.
  JOURNAL   Nat Methods. 2011 Dec 4. doi: 10.1038/nmeth.1802.
  PUBMED    22138822
REFERENCE   2  (bases 1 to 7889)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7889)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2011 Dec 4. doi: 10.1038/nmeth.1802."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7889
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..515
                     /label=MSCV
                     /note="Murine embryonic stem cell virus promoter including
                     the enhancer and promoter region of PCMV virus and 5' 
                     untranslated region of dl-587rev retrovirus"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell
                     virus"
     CDS             987..1403
                     /codon_start=1
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag
                     gene lacking the start codon"
                     /translation="GQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWPTF
                     NVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVHPKP
                     PPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGA"
     protein_bind    1437..1470
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             1505..2179
                     /codon_start=1
                     /label=DsRed-Express2
                     /note="noncytotoxic tetrameric variant of DsRed fluorescent
                     protein (Strack et al., 2008)"
                     /translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGVLKGEIHK
                     ALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQYERAEARHH
                     LFQ"
     protein_bind    complement(2207..2240)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2291..2317
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     CDS             2327..2353
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     CDS             2363..2389
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     promoter        2417..2916
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     CDS             2937..3533
                     /codon_start=1
                     /label=PuroR
                     /note="puromycin N-acetyltransferase"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     misc_feature    3606..4194
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     LTR             4251..4765
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem
                     cell virus"
     primer_bind     complement(4934..4950)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4958..4974)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(4982..5012)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5027..5048)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(5165..5182)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5336..5924)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6098..6955)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6956..7060)
                     /label=AmpR promoter
     primer_bind     7128..7146
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     primer_bind     complement(7184..7206)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7306..7325
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     7519..7541
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7669..7688
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI,
                     reverse primer"
     LTR             7888..7889
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaatta gatcactagt ccagtgtggt ggaattataa
     1441 cttcgtatag catacattat acgaagttat aattctgcag atatccagca cagtggcggc
     1501 caccatggat agcactgaga acgtcatcaa gcccttcatg cgcttcaagg tgcacatgga
     1561 gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggca agccctacga
     1621 gggcacccag accgccaagc tgcaggtgac caagggcggc cccctgccct tcgcctggga
     1681 catcctgtcc ccccagttcc agtacggctc caaggtgtac gtgaagcacc ccgccgacat
     1741 ccccgactac aagaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
     1801 cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcaccttcat
     1861 ctaccacgtg aagttcatcg gcgtgaactt cccctccgac ggccccgtaa tgcagaagaa
     1921 gactctgggc tgggagccct ccaccgagcg cctgtacccc cgcgacggcg tgctgaaggg
     1981 cgagatccac aaggcgctga agctgaaggg cggcggccac tacctggtgg agttcaagtc
     2041 aatctacatg gccaagaagc ccgtgaagct gcccggctac tactacgtgg actccaagct
     2101 ggacatcacc tcccacaacg aggactacac cgtggtggag cagtacgagc gcgccgaggc
     2161 ccgccaccac ctgttccagt agagctagct gaataaggcc gctcgaataa cttcgtatag
     2221 catacattat acgaagttat tcgagtctag agggcccgtt agatctctcg aggttaacga
     2281 attcgactac tacccctacg acgtgcccga ctacgccgct ggagcatacc cctacgacgt
     2341 gcccgactac gccgctggag cataccccta cgacgtgccc gactacgcct gagtcgacgc
     2401 ggccgccaat tctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct
     2461 ttagcagccc cgctgggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc
     2521 acatccaccg gtaggcgcca accggctccg ttctttggtg gccccttcgc gccaccttct
     2581 actcctcccc tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg caggacgtga
     2641 caaatggaag tagcacgtct cactagtctc gtgcagatgg acagcaccgc tgagcaatgg
     2701 aagcgggtag gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct
     2761 cagaggctgg gaaggggtgg gtccgggggc gggctcaggg gcgggctcag gggcggggcg
     2821 ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc
     2881 gcgctgttct cctcttcctc atctccgggc ctttcgacct gcagcccaag cttaccatga
     2941 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca
     3001 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc
     3061 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     3121 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     3181 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     3241 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     3301 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg
     3361 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     3421 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     3481 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgacgcc
     3541 cgccccacga cccgcagcgc ccgaccgaaa ggagcgcacg accccatgca tcgataattc
     3601 ccgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     3661 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     3721 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     3781 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     3841 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     3901 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     3961 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
     4021 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     4081 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     4141 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcggg
     4201 aattatcgat aaaataaaag attttattta gtctccagaa aaagggggga atgaaagacc
     4261 ccacctgtag gtttggcaag ctagcttaag taacgccatt ttgcaaggca tggaaaatac
     4321 ataactgaga atagagaagt tcagatcaag gttaggaaca gagagacagc agaatatggg
     4381 ccaaacagga tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt
     4441 ccccagatgc ggtcccgccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc
     4501 cccaaggacc tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc
     4561 ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg
     4621 cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tgtatccaat aaaccctctt
     4681 gcagttgcat ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga
     4741 ctacccgtca gcgggggtct ttcatgggta acagtttctt gaagttggag aacaacattc
     4801 tgagggtagg agtcgaatat taagtaatcc tgactcaatt agccactgtt ttgaatccac
     4861 atactccaat actcctgaaa tagttcatta tggacagcgc agaagagctg gggagaatta
     4921 attcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4981 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     5041 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5101 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5161 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5221 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5281 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5341 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5401 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5461 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5521 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5581 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5641 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5701 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5761 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5821 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5881 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5941 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6001 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6061 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6121 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6181 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6241 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6301 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6361 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6421 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6481 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6541 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6601 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6661 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6721 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6781 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6841 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6901 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6961 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     7021 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7081 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     7141 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     7201 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     7261 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
     7321 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     7381 accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     7441 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
     7501 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggcgcaa ggaatggtgc
     7561 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     7621 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     7681 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     7741 gtagaggcga ttagtccaat ttgttaaaga caggatatca gtggtccagg ctctagtttt
     7801 gactcaacaa tatcaccagc tgaagcctat agagtacgag ccatagataa aataaaagat
     7861 tttatttagt ctccagaaaa aggggggaa
//
