LOCUS       sequence-10176-e        7474 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7474)
  AUTHORS   Hockemeyer D, Soldner F, Beard C, Gao Q, Mitalipova M, Dekelver RC, 
            Katibah GE, Amora R, Boydston EA, Zeitler B, Meng X, Miller JC, 
            Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R
  TITLE     Efficient targeting of expressed and silent genes in human ESCs and 
            iPSCs using zinc-finger nucleases.
  JOURNAL   Nat Biotechnol. 2009 Sep;27(9):851-7. doi: 10.1038/nbt.1562
  PUBMED    19680244
REFERENCE   2  (bases 1 to 7474)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nbt"; 
            journalName: "Nat Biotechnol"; date: "2009-09"; volume: "27"; issue:
            "9"; pages: "851-7"
COMMENT     Sequence Label: OCT4 GFP puro Donor 1
FEATURES             Location/Qualifiers
     source          1..7474
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        242..260
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    1064..1089
                     /label=SA
                     /note="splice acceptor site"
     CDS             join(1092..1094,1095..1097,1098..1808)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1092..1094,1095..1097,1098..1808)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1137..1158)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1398..1417)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1745..1766
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1935..2534
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1935..1954)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2431..2451
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3846..3865)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3847..3865)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3872..3889)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3872..3888)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3881..3903)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             4026..4328
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     4207..4226
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             4677..5471
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4677..5471
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4731..4750)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5341..5360
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             complement(join(5721..6512,6513..6581))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6344..6363
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6705..7293
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7194..7213
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7447..7464
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctca
      241 gaattaaccc tcactaaagg gactagtcct gcaggtttaa acgaattcgc ccttgacctg
      301 cactgaggtc ctggaggggc gccagttgtg tctcccggtt ttccccttcc acagacacca
      361 ttgccaccac cattaggcaa acatccttcg cctcagtttc tccccccacc tccctctcct
      421 ccacccatcc agggggcggg gccagaggtc aaggctagtg ggtgggactg gggagggaga
      481 gaggggttga gtagtccctt cgcaagccct catttcacca ggcccccggc ttggggcgcc
      541 ttccttcccc atggcgggac acctggcttc ggatttcgcc ttctcgcccc ctccaggtgg
      601 tggaggtgat gggccagggg ggccggagcc gggctgggtt gatcctcgga cctggctaag
      661 cttccaaggc cctcctggag ggccaggaat cgggccgggg gttgggccag gctctgaggt
      721 gtgggggatt cccccatgcc ccccgccgta tgagttctgt ggggggatgg cgtactgtgg
      781 gccccaggtt ggagtggggc tagtgcccca aggcggcttg gagacctctc agcctgaggg
      841 cgaagcagga gtcggggtgg agagcaactc cgatggggcc tccccggagc cctgcaccgt
      901 cacccctggt gccgtgaagc tggagaagga gaagctggag caaaacccgg aggaggcaag
      961 tgagcttcga cggggttggg gtgtggggag gtggtcatga cagggcagcc tgatggggaa
     1021 gtggtcacct gcagctgccc agacctggca cccaggagga tccctgacct cttctcttcc
     1081 tcccacaggc catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     1141 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     1201 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     1261 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     1321 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     1381 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     1441 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     1501 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     1561 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     1621 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     1681 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     1741 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     1801 tgtacaagta cagcggtcgc gactctagag tcgacggttc tggtaagcaa actttgaatt
     1861 ttgaccttct taagcttgcg ggagacgtcg agtccaaccc cgggcccgca tgcaagcttc
     1921 agctgaagct taccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg
     1981 tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca
     2041 ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc
     2101 gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct
     2161 ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg
     2221 ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc
     2281 accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg
     2341 gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg
     2401 tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct
     2461 tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca
     2521 agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac
     2581 cccatgcatc gatgatatca gatccccggg atgcagaaat tgatgatcta ttaaacaata
     2641 aagatgtcca ctaaaatgga agtttttcct gtcatacttt gttaagaagg gtgagaacag
     2701 agtacctaca ttttgaatgg aaggattgga gctacggggg tgggggtggg gtgggattag
     2761 ataaatgcct gctctttact gaaggctctt tactattgct ttatgataat gtttcatagt
     2821 tggatatcat aatttaaaca agcaaaacca aattaagggc cagctcattc ctcccactca
     2881 tgatctatag atctatagat ctctcgtggg atcattgttt ttctcttgat tcccactttg
     2941 tggttctaag tactgtggtt tccaaatgtg tcagtttcat agcctgaaga acgagatcag
     3001 cagcctctgt tccacataca cttcattctc agtattgttt tgccaagttc taattccatc
     3061 agaagctggt cgaggatccg aggagcaggc agggtcagct gccctggcca gggaggggtg
     3121 tgtatcaact gcaggcagcc ctggcaggca ggggccaggt gggaactgga agctggattt
     3181 cgaagagaca actgccggtg agggcagagc ggcctgggag agtcagaagc tggcccaggc
     3241 tggcctttgc tctggcccag cccttgtcag ggtctctcac atctcctagg cctgcccagg
     3301 gtctggtcac tcattactgg cccagcacca gacccaggtt ggggttggtt tgagcccctt
     3361 ttcccaccct tagtcctgct tgaaaatttg acccttatca gacccaagat tttggcctta
     3421 gggttaagca tagcctgagg gtaaaaacag tgctcattcc aggattattg ttcctgaaag
     3481 tctagggtgt gactcgtttc tgataggatc tcctgtttgg gctgtgtgtg tgcgcgttgt
     3541 gagctgggtt tacctccagt caagtatagg gcttgtcttc cccggatctc tgcctcaggc
     3601 caatgactgg ccactgtgtt aaggtgcaca ccctggcacc ccttgtagaa agctggattt
     3661 tgattgactt cagcctcagt tccaaagttg taaacaagaa aaatggtgag agatttctcc
     3721 aggccatttg caaatataga gctgctgcgg gattgaaggc atccagccct gctgaggact
     3781 attaaagatg tatcttccag tccttcaagg cgaagggcga attcgcggcc gctaaattca
     3841 attcgcccta tagtgagtcg tattacaatt cactggccgt cgttttacaa cgtcgtgact
     3901 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     3961 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctatacg
     4021 tacggcagtt taaggtttac acctataaaa gagagagccg ttatcgtctg tttgtggatg
     4081 tacagagtga tattattgac acgccggggc gacggatggt gatccccctg gccagtgcac
     4141 gtctgctgtc agataaagtc tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa
     4201 gctggcgcat gatgaccacc gatatggcca gtgtgccggt ctccgttatc ggggaagaag
     4261 tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg
     4321 gaatataaat gtcaggcatg agattatcaa aaaggatctt cacctagatc cttttcacgt
     4381 agaaagccag tccgcagaaa cggtgctgac cccggatgaa tgtcagctac tgggctatct
     4441 ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc
     4501 gatagctaga ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc
     4561 cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga
     4621 tctgatggcg caggggatca agctctgatc aagagacagg atgaggatcg tttcgcatga
     4681 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     4741 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     4801 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag
     4861 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     4921 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     4981 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     5041 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     5101 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     5161 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg
     5221 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     5281 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     5341 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     5401 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     5461 agttcttctg aattattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc
     5521 tgtgcggtat ttcacaccgc atcaggtggc acttttcggg gaaatgtgcg cggaacccct
     5581 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagatt atcaaaaagg
     5641 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5701 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5761 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5821 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5881 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5941 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6001 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6061 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6121 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6181 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6241 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6301 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6361 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6421 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6481 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6541 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6601 tattgaagca tttatcaggg ttattgtctc atgaccaaaa tcccttaacg tgagttttcg
     6661 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6721 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6781 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6841 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6901 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6961 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     7021 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     7081 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     7141 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     7201 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     7261 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     7321 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     7381 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     7441 gagcgcagcg agtcagtgag cgaggaagcg gaag
//
