LOCUS       62056_13465        6794 bp DNA     circular SYN 15-FEB-2020
DEFINITION  Expression vector pKK44, complete sequence.
ACCESSION   MN811105
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6794)
  AUTHORS   Krawczyk K, Scheller L, Kim H, Fussenegger M.
  TITLE     Rewiring of endogenous signaling pathways to genomic targets for 
            therapeutic cell reprogramming
  JOURNAL   Nat Commun 11 (1), 608 (2020)
  PUBMED    32001704
REFERENCE   2  (bases 1 to 6794)
  AUTHORS   Krawczyk KK.
  TITLE     Direct Submission
  JOURNAL   Submitted (09-DEC-2019) D-BSSE, ETH Zurich, Mattenstrasse 26, Basel 
            4058, Switzerland
REFERENCE   3  (bases 1 to 6794)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun"; date: "2020"; volume: "11"; issue: "1"; pages: "608"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (09-DEC-2019) D-BSSE, ETH Zurich, Mattenstrasse 26, Basel 4058, 
            Switzerland"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..6794
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    21..245
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      291..719
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        733..1062
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             1129..1920
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    2097..2230
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2267..2283)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2291..2307)
                     /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(2315..2345)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(2360..2381)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(2669..3254)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3428..4285)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4286..4390)
                     /label=AmpR promoter
     enhancer        4656..5035
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5036..5239
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        5284..5302
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5353..5739
                     /codon_start=1
                     /label=MS2-N55K
                     /note="bacteriophage MS2 coat protein"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             5794..6336
                     /codon_start=1
                     /label=p65
                     /note="C-terminal portion of the p65 subunit of mouse
                     NF-kappa-B"
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             6361..6732
                     /codon_start=1
                     /label=HSF1
                     /note="C-terminal activation domain from the human heat
                     shock transcription factor HSF1"
                     /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
                     PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
                     PTISLLTGSEPPKAKDPTVS"
ORIGIN
        1 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
       61 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
      121 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
      181 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
      241 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
      301 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
      361 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
      421 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
      481 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
      541 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
      601 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
      661 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
      721 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
      781 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
      841 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
      901 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
      961 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
     1021 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
     1081 atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     1141 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     1201 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     1261 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     1321 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     1381 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     1441 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     1501 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     1561 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     1621 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     1681 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     1741 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     1801 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     1861 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     1921 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     1981 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     2041 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     2101 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     2161 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     2221 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     2281 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     2341 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     2401 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     2461 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     2521 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     2581 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     2641 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     2701 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     2761 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     2821 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     2881 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     2941 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     3001 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     3061 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     3121 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     3181 gcaaacaaac caccgctggt agcggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     3241 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     3301 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     3361 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     3421 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     3481 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     3541 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     3601 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3661 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3721 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3781 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3841 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3901 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3961 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     4021 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     4081 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     4141 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     4201 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     4261 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     4321 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     4381 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
     4441 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     4501 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
     4561 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
     4621 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
     4681 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     4741 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     4801 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     4861 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     4921 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     4981 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
     5041 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
     5101 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
     5161 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
     5221 ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga
     5281 aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttcgt
     5341 acggccacca tggcttcaaa ctttactcag ttcgtgctcg tggacaatgg tgggacaggg
     5401 gatgtgacag tggctccttc taatttcgct aatggggtgg cagagtggat cagctccaac
     5461 tcacggagcc aggcctacaa ggtgacatgc agcgtcaggc agtctagtgc ccagaagaga
     5521 aagtatacca tcaaggtgga ggtccccaaa gtggctaccc agacagtggg cggagtcgaa
     5581 ctgcctgtcg ccgcttggag gtcctacctg aacatggagc tcactatccc aattttcgct
     5641 accaattctg actgtgaact catcgtgaag gcaatgcagg ggctcctcaa agacggtaat
     5701 cctatccctt ccgccatcgc cgctaactca ggtatctaca gcgctggagg aggtggaagc
     5761 ggaggaggag gaagcggagg aggaggtagc ggaccttcag ggcagatcag caaccaggcc
     5821 ctggctctgg cccctagctc cgctccagtg ctggcccaga ctatggtgcc ctctagtgct
     5881 atggtgcctc tggcccagcc acctgctcca gcccctgtgc tgaccccagg accaccccag
     5941 tcactgagcg ctccagtgcc caagtctaca caggccggcg aggggactct gagtgaagct
     6001 ctgctgcacc tgcagttcga cgctgatgag gacctgggag ctctgctggg gaacagcacc
     6061 gatcccggag tgttcacaga tctggcctcc gtggacaact ctgagtttca gcagctgctg
     6121 aatcagggcg tgtccatgtc tcatagtaca gccgaaccaa tgctgatgga gtaccccgaa
     6181 gccattaccc ggctggtgac cggcagccag cggccccccg accccgctcc aactcccctg
     6241 ggaaccagcg gcctgcctaa tgggctgtcc ggagatgaag acttctcaag catcgctgat
     6301 atggacttta gtgccctgct gtcacagatt tcctctagtg ggcagggagg aggtggaagc
     6361 ggcttcagcg tggacaccag tgccctgctg gacctgttca gcccctcggt gaccgtgccc
     6421 gacatgagcc tgcctgacct tgacagcagc ctggccagta tccaagagct cctgtctccc
     6481 caggagcccc ccaggcctcc cgaggcagag aacagcagcc cggattcagg gaagcagctg
     6541 gtgcactaca cagcgcagcc gctgttcctg ctggaccccg gctccgtgga caccgggagc
     6601 aacgacctgc cggtgctgtt tgagctggga gagggctcct acttctccga aggggacggc
     6661 ttcgccgagg accccaccat ctccctgctg acaggctcgg agcctcccaa agccaaggac
     6721 cccactgtct cctgtacaga attctgcaga tatccagcac agtggcggcc gctcgagtct
     6781 agagggcccg ttta
//
