LOCUS       62056_13480        6182 bp DNA     circular SYN 15-FEB-2020
DEFINITION  Expression vector pKK82, complete sequence.
ACCESSION   MN811101
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6182)
  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 6182)
  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 6182)
  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..6182
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        242..621
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        622..825
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        870..888
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             939..1325
                     /codon_start=1
                     /label=MS2-N55K
                     /note="bacteriophage MS2 coat protein"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     polyA_signal    1789..2013
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2059..2487
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2501..2830
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             2897..3688
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3865..3998
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4035..4051)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4059..4075)
                     /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(4083..4113)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4128..4149)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(4437..5022)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5196..6053)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6054..6158)
                     /label=AmpR promoter
ORIGIN
        1 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
       61 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
      121 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag
      181 aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt
      241 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
      301 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      361 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      421 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      481 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      541 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      601 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
      661 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
      721 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
      781 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga
      841 gaacccactg cttactggct tatcgaaatt aatacgactc actataggga gacccaagct
      901 ggctagcgtt taaacttaag cttcgtacgg ccaccatggc ttcaaacttt actcagttcg
      961 tgctcgtgga caatggtggg acaggggatg tgacagtggc tccttctaat ttcgctaatg
     1021 gggtggcaga gtggatcagc tccaactcac ggagccaggc ctacaaggtg acatgcagcg
     1081 tcaggcagtc tagtgcccag aagagaaagt ataccatcaa ggtggaggtc cccaaagtgg
     1141 ctacccagac agtgggcgga gtcgaactgc ctgtcgccgc ttggaggtcc tacctgaaca
     1201 tggagctcac tatcccaatt ttcgctacca attctgactg tgaactcatc gtgaaggcaa
     1261 tgcaggggct cctcaaagac ggtaatccta tcccttccgc catcgccgct aactcaggta
     1321 tctacagcgc tggaggaggt ggaagcggag gaggaggaag cggaggagga ggtagcggac
     1381 cgatctccca gccgcagaag ggccggaagc cccgggacct agagcttcca ctcagcccga
     1441 gcctgctagg tgggccggga cccgaacgga ccccaggatc gggaagtggc tccggcctcc
     1501 aggctccggg gccggcgctg accccatccc tgcttcctac gcatacattg accccggtgc
     1561 tgctgacacc cagctcgctg cctcctagca ttcacttctg gagcaccctg agtcccattg
     1621 cgccccgtag cccggccaag ctctccttcc agtttccatc cagtggcagc gcccaggtgc
     1681 acatcccttc tatcagcgtg gatggcctct cgacccccgt ggtgctctcc ccagggcccc
     1741 agaagccatg atctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta
     1801 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     1861 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     1921 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     1981 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     2041 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2101 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2161 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     2221 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     2281 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2341 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     2401 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     2461 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     2521 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2581 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2641 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     2701 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     2761 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     2821 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg
     2881 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     2941 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     3001 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     3061 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     3121 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     3181 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     3241 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     3301 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     3361 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     3421 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     3481 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     3541 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     3601 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     3661 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     3721 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     3781 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     3841 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     3901 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     3961 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     4021 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     4081 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     4141 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4201 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4261 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4321 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4381 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4441 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4501 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4561 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4621 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4681 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4741 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4801 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4861 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4921 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggttttttt
     4981 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5041 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5101 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5161 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5221 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5281 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5341 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5401 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5461 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     5521 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     5581 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     5641 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     5701 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     5761 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     5821 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     5881 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     5941 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6001 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6061 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6121 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6181 cc
//
