LOCUS       62056_13470        9065 bp DNA     circular SYN 15-FEB-2020
DEFINITION  Expression vector pKK50, complete sequence.
ACCESSION   MN811100
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9065)
  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 9065)
  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 9065)
  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..9065
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(247..832)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1006..1863)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(1864..1968)
                     /label=AmpR promoter
     enhancer        2234..2613
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2614..2817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        2862..2880
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    2928..3325
                     /gene="GEAR-NFAT"
                     /label=MS2
                     /note="MS2"
     CDS             4371..4397
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
     CDS             4401..4427
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
     CDS             4434..4460
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
     misc_feature    5739..5762
                     /gene="GEAR-NFAT"
                     /label=DNA binding
                     /note="DNA binding"
     CDS             5871..6587
                     /label=EGFP
                     /note="enhanced GFP"
     polyA_signal    6664..6888
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6934..7362
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        7376..7705
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             7772..8563
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     polyA_signal    8740..8873
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8910..8926)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(8934..8950)
                     /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(8958..8988)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(9003..9024)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
ORIGIN
        1 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
       61 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
      121 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
      181 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
      241 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
      301 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
      361 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
      421 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
      481 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
      541 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
      601 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
      661 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
      721 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
      781 cggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
      841 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
      901 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
      961 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     1021 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     1081 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     1141 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     1201 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     1261 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     1321 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     1381 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     1441 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     1501 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     1561 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     1621 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     1681 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     1741 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     1801 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     1861 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     1921 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     1981 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc
     2041 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg
     2101 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
     2161 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
     2221 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
     2281 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     2341 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     2401 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     2461 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     2521 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
     2581 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
     2641 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     2701 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     2761 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc
     2821 tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg
     2881 gagacccaag ctggctagcg tttaaactta agcttcgtac ggccaccatg gcttcaaact
     2941 ttactcagtt cgtgctcgtg gacaatggtg ggacagggga tgtgacagtg gctccttcta
     3001 atttcgctaa tggggtggca gagtggatca gctccaactc acggagccag gcctacaagg
     3061 tgacatgcag cgtcaggcag tctagtgccc agaagagaaa gtataccatc aaggtggagg
     3121 tccccaaagt ggctacccag acagtgggcg gagtcgaact gcctgtcgcc gcttggaggt
     3181 cctacctgaa catggagctc actatcccaa ttttcgctac caattctgac tgtgaactca
     3241 tcgtgaaggc aatgcagggg ctcctcaaag acggtaatcc tatcccttcc gccatcgccg
     3301 ctaactcagg tatctacagc gctggaggag gtggaagcgg aggaggagga agcggaggag
     3361 gaggtagcgg accttcaggg cagatcagca accaggccct ggctctggcc cctagctccg
     3421 ctccagtgct ggcccagact atggtgccct ctagtgctat ggtgcctctg gcccagccac
     3481 ctgctccagc ccctgtgctg accccaggac caccccagtc actgagcgct ccagtgccca
     3541 agtctacaca ggccggcgag gggactctga gtgaagctct gctgcacctg cagttcgacg
     3601 ctgatgagga cctgggagct ctgctgggga acagcaccga tcccggagtg ttcacagatc
     3661 tggcctccgt ggacaactct gagtttcagc agctgctgaa tcagggcgtg tccatgtctc
     3721 atagtacagc cgaaccaatg ctgatggagt accccgaagc cattacccgg ctggtgaccg
     3781 gcagccagcg gccccccgac cccgctccaa ctcccctggg aaccagcggc ctgcctaatg
     3841 ggctgtccgg agatgaagac ttctcaagca tcgctgatat ggactttagt gccctgctgt
     3901 cacagatttc ctctagtggg cagggaggag gtggaagcgg cttcagcgtg gacaccagtg
     3961 ccctgctgga cctgttcagc ccctcggtga ccgtgcccga catgagcctg cctgaccttg
     4021 acagcagcct ggccagtatc caagagctcc tgtctcccca ggagcccccc aggcctcccg
     4081 aggcagagaa cagcagcccg gattcaggga agcagctggt gcactacaca gcgcagccgc
     4141 tgttcctgct ggaccccggc tccgtggaca ccgggagcaa cgacctgccg gtgctgtttg
     4201 agctgggaga gggctcctac ttctccgaag gggacggctt cgccgaggac cccaccatct
     4261 ccctgctgac aggctcggag cctcccaaag ccaaggaccc cactgtctcc tgtacaggcg
     4321 gaagcggcgg ttcaggaggc agcggtggat caggctccac catgatcttt tacccatacg
     4381 atgttcctga ctatgcgggc tatccctatg acgtcccgga ctatgcagga tcctatccat
     4441 atgacgttcc agattacgct gctcagtgcg gccgctctag cccggagccg cagcccgacc
     4501 ccgatggcgg ggacggcccc ggccacgagc ccgggggcag tccccaagac gagctggact
     4561 tttccatcct cttcgattat gactatctga accctatcga agaagaaccg atcgcacata
     4621 aggccatcag ctcaccctcc ggactcgcat acccggatga tgtcctggac tatggcctca
     4681 agccatgcaa cccccttgcc agtccctctg gcgagccccc tggccggttc ggagagccgg
     4741 atagtatagg gttccagaac tttctgagcc cggtcaagcc agcaggggct tcgggcccga
     4801 gccctcggat cgagatcact ccatcccacg aactgatgca ggcagggggg gccctccgtg
     4861 ggagagacgc cggcctgtcc cccgagcagc cggccctggc cctggccggc gtggccgcca
     4921 gcccgaggtt cacactgccc gtgcccggct acgagggcta ccgcgagccg ctttgcttga
     4981 gccccgctag cagcggctcc tctgccagct tcatttctga caccttctcc ccctacacct
     5041 cgccctgcgt ctcacccaat aacgccgggc ccgacgacct gtgtccccag tttcaaaaca
     5101 tccctgctca ttattccccc agaacctctc caataatgtc acctcgaacc agcctcgccg
     5161 aggacagctg cctgggccga cactcgcccg tgccccgtcc ggcatcccgc tcctcctcac
     5221 ccggtgccaa gcggaggcat tcgtgcgcag aggctttggt tgctcctctg cccgcagcct
     5281 caccccagcg ctcccggagc ccctcgccac agccctcgcc tcacgtggca ccgcaggacg
     5341 acagcatccc cgctgggtac ccccccacgg ccggctctgc tgttctcatg gatgccctca
     5401 acaccctggc caccgactcg ccctgcggga tcccctccaa gatatggaag accagtcctg
     5461 acccgacgcc tgtgtccacc gctccgtcca aggctggcct ggcccgccac atctacccta
     5521 ctgtggagtt cctggggcca tgtgagcagg aggagaggag gaattccgct ccagagtcca
     5581 tcctgctggt accacctact tggcccaagc agttggtgcc ggccattccc atctgcagca
     5641 tccctgtgac tgcatccctc ccaccactcg agtggccact ctccaatcag tcgggctcct
     5701 atgagctacg gattgaggtc caacccaagc cccatcaccg ggcccactat gagacggagg
     5761 gcagccgtgg cgctgtcaaa gccccaacag gaggacaccc tgtggtgcag ctccacggct
     5821 acatggagaa caagcctctg gggcttcaga tcgatccacc ggtcgccacc atggtgagca
     5881 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     5941 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     6001 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     6061 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     6121 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     6181 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     6241 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     6301 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     6361 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     6421 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     6481 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     6541 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggaa ttctgcagat
     6601 atccagcaca gtggcggccg ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct
     6661 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     6721 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     6781 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     6841 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     6901 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     6961 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     7021 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     7081 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     7141 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     7201 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     7261 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     7321 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
     7381 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     7441 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     7501 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     7561 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     7621 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     7681 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
     7741 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     7801 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     7861 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     7921 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     7981 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     8041 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     8101 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     8161 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     8221 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     8281 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     8341 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     8401 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     8461 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     8521 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     8581 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     8641 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     8701 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     8761 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     8821 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     8881 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     8941 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     9001 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     9061 aacct
//
