LOCUS       pET28a-MH6-RspCa        9416 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses E. coli codon optimized RspCas13d and RspCasWYL1 in the 
            pET28a backbone..
ACCESSION   .
VERSION     .
KEYWORDS    pET28a-MH6-RspCas13d_RspCasWYL1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9416)
  AUTHORS   Yan WX, Chong S, Zhang H, Makarova KS, Koonin EV, Cheng DR, Scott DA
  TITLE     Cas13d Is a Compact RNA-Targeting Type VI CRISPR Effector Positively
            Modulated by a WYL-Domain-Containing Accessory Protein.
  JOURNAL   Mol Cell. 2018 Mar 9. pii: S1097-2765(18)30173-4. doi: 
            10.1016/j.molcel.2018.02.028.
  PUBMED    29551514
REFERENCE   2  (bases 1 to 9416)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9416)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Cell. 
            2018 Mar 9. pii: S1097-2765(18)30173-4. doi: 
            10.1016/j.molcel.2018.02.028."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9416
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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        53..82
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     RBS             102..124
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             156..173
                     /label=6xHis
                     /note="6xHis affinity tag"
     RBS             2956..2978
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3010..3027
                     /label=6xHis
                     /note="6xHis affinity tag"
     promoter        4222..4256
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological
                     Parts BBa_J23119)"
     terminator      4365..4412
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4449..4904
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(4999..5811)
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     rep_origin      5933..6521
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6675..6692
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    complement(6707..6846)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6932..6954
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6951..7139)
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     protein_bind    complement(7914..7935)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(7951..9030)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(9031..9108)
                     /label=lacI promoter
     primer_bind     9314..9333
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI,
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gatttacact
       61 ttatgcttcc ggctcgtatg tttctagaga attcaaataa ttttgtttaa ctttaagaag
      121 gagatttaaa tatgaaaatc gaagaaggta aaggtcacca tcaccatcac cacatggcca
      181 aaaagaacaa aatgaaaccg cgtgaactgc gtgaagcaca gaaaaaagca cgtcagctga
      241 aagcagcaga aattaacaat aatgcagcac cggcaattgc agcaatgcct gcagcagaag
      301 ttattgcacc ggttgcagaa aaaaagaaaa gcagcgttaa agcagccggt atgaaaagca
      361 ttctggtgag caaaaacaaa atgtatatca ccagctttgg caaaggtaat agcgcagttc
      421 tggaatatga agtggataac aacgattaca atcagaccca gctgagcagc aaaggtagca
      481 gcaatattga gctgcgtggt gttaatgaag tgaacattac ctttagcagc aaacacggtt
      541 ttgaaagcgg tgttgaaatc aataccagca atccgacaca tcgtagcggt gaaagcagtc
      601 cggttcgtgg tgatatgctg ggcctgaaaa gcgaactgga aaaacgcttt tttggcaaaa
      661 ccttcgatga caacattcat atccagctga tctataacat cctggacatc gaaaaaattc
      721 tggccgttta tgtgaccaac attgtgtatg cactgaataa catgctgagc attaaagata
      781 gcgagagcta tgatgatttc atgggttatc tgagcgcacg caatacctat gaagttttta
      841 cccatccgga taaaagcaac ctgagcgata aagcaaaagg caacatcaaa aaatccttta
      901 gcaccttcaa cgacctgctg aaaaccaaac gtctgggtta ttttggtctg gaagaaccga
      961 aaacaaaaga tacccgtgtt agccaggcat acaaaaaacg tgtttatcac atgctggcaa
     1021 ttgtgggtca gattcgtcag agcgtgtttc atgataaaag tagcaaactg gatgaggacc
     1081 tgtatagctt tatcgatatc atcgattccg aatatcgtga aaccctggat tatctggttg
     1141 atgaacgttt tgacagcatc aacaaaggtt ttatccaggg caacaaagtg aatattagcc
     1201 tgctgatcga tatgatgaaa ggctatgaag ccgatgatat tatccgcctg tattatgatt
     1261 ttatcgtgct gaaaagccag aagaatctgg gctttagtat caaaaaactg cgcgagaaaa
     1321 tgctggatga atatggcttt cgcttcaaag ataaacagta tgatagcgtg cgtagcaaga
     1381 tgtataaact gatggatttt ctgctgttct gcaactatta tcgcaatgat gttgttgccg
     1441 gtgaagcact ggttcgtaaa ctgcgtttta gcatgaccga tgatgaaaaa gaaggtattt
     1501 acgcagacga agcaagcaaa ctgtggggta aatttcgtaa cgattttgag aacattgccg
     1561 accatatgaa cggtgatgtt attaaagaac tgggcaaagc cgatatggat ttcgatgaaa
     1621 aaatactgga cagcgagaaa aaaaacgcaa gcgatctgct gtatttcagc aagatgattt
     1681 atatgctgac ctatttcctg gatggcaaag aaattaatga tctgctgacc acgctgatta
     1741 gcaaatttga taacatcaaa gaatttctga aaatcatgaa aagctcagcc gttgatgttg
     1801 aatgtgaact gaccgcaggt tataaactgt ttaatgatag ccagcgcatt accaacgaac
     1861 tgttcattgt taaaaacatt gcgagcatgc gtaaaccggc aagcagcgca aaactgacca
     1921 tgtttcgtga tgcactgacc attctgggta ttgatgataa cattaccgat gatcgcatta
     1981 gcgaaatcct gaagctgaaa gaaaaaggta aaggtattca tggcctgcgc aactttatta
     2041 ccaataacgt gattgaaagc tcccgctttg tgtacctgat caaatatgca aatgcccaga
     2101 aaattcgcaa agtggcgaaa aatgaaaagg tggtgatgtt tgtgttaggt ggtattccgg
     2161 atacacagat tgaacgctat tacaaaagct gcgttgaatt tccggatatg aacagcagcc
     2221 tggaagttaa acgtagtgaa ctggcacgta tgatcaaaaa catcagcttc gacgatttca
     2281 agaacgttaa acagcaggca aaaggtcgtg aaaatgttgc aaaagaacgt gccaaagcag
     2341 tgattggtct gtatctgacc gttatgtatc tgctggttaa aaatctggtt aacgtgaatg
     2401 cccgttatgt gattgcaatt cattgtctgg aacgtgattt cggcctgtat aaagaaatca
     2461 ttccggaact ggcaagcaag aacctgaaaa atgattatcg tattctgagt cagaccctgt
     2521 gtgaactgtg tgataaaagc ccgaacctgt ttctgaagaa aaacgaacgt ctgcgcaaat
     2581 gcgttgaggt ggatattaac aatgcagata gcagcatgac ccgcaaatat cgtaattgta
     2641 ttgcacatct gacagttgtg cgcgaactga aagaatatat tggtgatatt cgtaccgtgg
     2701 acagctactt tagcatctat cattatgtta tgcagcggtg tattaccaaa cgcgaaaatg
     2761 ataccaaaca agaagagaaa atcaaatacg aggacgatct gcttaaaaac cacggctata
     2821 ccaaagattt tgtgaaagcc ctgaatagcc cgttcggtta taacattccg cgtttcaaaa
     2881 atctgagcat cgagcagctg tttgatcgta atgaatatct gacggaaaaa taataagaat
     2941 tcggatccaa ataattttgt ttaactttaa gaaggagatt taaatatgaa aatcgaagaa
     3001 ggtaaaggtc accatcacca tcaccacatg ctgattccgc ctagcacctt tctgccgaaa
     3061 cgtgataaaa atgttccgta tattgcagag gttcagagca ttccgctgag cccgagcgca
     3121 tatagcgtta ttatcaaaga taagagcatc tttgaaacca gcctgagtcc gaatggtagc
     3181 gttagcatga gcagctttct gaccagcatt tttgatagcg cctatatcgc aagcctgaaa
     3241 tacaaaagcg acgacaacta taaatacatt ggtattccgc tgctgaacgc ctttgttaaa
     3301 tggcagattg aagaaattga tgatggcctg gatgacaaga gcaaagaaat cattaaaagc
     3361 tatctgatca gcaagctgag cgccaaatat gaaaaaacca aaaccgaaaa tgccgttcgt
     3421 gttcgtctga gcatttgtcg tgatctgtat gataccctga gcagcgacga tctgtattat
     3481 gaaaataaag tgtatagcag caccctgcgt cgttttctga aagcagttta tgaagattat
     3541 gcactgctga gcgattgtga acgtgaacgt ctgatttttg ccgataacat catcaaaatc
     3601 aacgaagtga ttaagcagaa tggcagccgt tattatagct tcatttatgc ctatagcaac
     3661 atgtacagcc gtgaaaaacg tcgtattcgc ctgattccgt atcgtattgt ttccgatgag
     3721 tacaaaatgt ataactacct ggtttgtctg agcgacgaaa aaagcgcagg taaagaattt
     3781 aaagcagata gctgtcgtat tagccgtctg agcggtctga gtattgcaga aaaactgagc
     3841 cagaaagaat atagcagcgt gaccgaatat gaacgcctga aagaagttca cgttaaaagc
     3901 gttaaacacc tgttaagcga tccgcgtttt ggtagtgatg aaagcgatat tagcaaagtg
     3961 tatctgaccg aaaaaggcgt ggaaatgttt ggcaaaattc tgtatcagcg tccgattctg
     4021 aaaggcaatg agaaaccgaa acctaatgcc gtgaatgaat ttatcagccc tccgattcag
     4081 gtgaaatact actttaacaa attcggcaaa gacggcgtta ttctgagccc gtctgatagc
     4141 tttgaagaga tgcgtaccct gtatgttgaa ggtgccgaag catataatcg tgaagttgaa
     4201 atgtaataag gatccctcga gttgacagct agctcagtcc taggtataat gctagcagag
     4261 accatggcca ccggtctctg ctaaagatat gaggagctca aactcgagct gctaacaaag
     4321 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     4381 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     4441 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     4501 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     4561 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4621 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     4681 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     4741 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     4801 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     4861 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     4921 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     4981 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     5041 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     5101 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     5161 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     5221 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     5281 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     5341 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     5401 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     5461 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     5521 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     5581 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     5641 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     5701 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     5761 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     5821 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     5881 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     5941 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6001 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6061 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     6121 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     6181 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6241 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6301 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     6361 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     6421 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     6481 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     6541 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     6601 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     6661 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     6721 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     6781 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     6841 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     6901 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     6961 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     7021 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     7081 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     7141 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     7201 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     7261 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     7321 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     7381 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     7441 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     7501 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     7561 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
     7621 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     7681 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     7741 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     7801 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     7861 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     7921 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     7981 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     8041 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     8101 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     8161 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     8221 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     8281 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     8341 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     8401 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     8461 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     8521 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     8581 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     8641 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     8701 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     8761 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     8821 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     8881 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     8941 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     9001 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     9061 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     9121 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     9181 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     9241 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     9301 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     9361 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaat
//
