LOCUS       sequence-10177-e        7549 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 7549)
  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 7549)
  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 2
FEATURES             Location/Qualifiers
     source          1..7549
                     /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"
     CDS             complement(1604..2203)
                     /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(1687..1707)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     2184..2203
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(join(2330..3040,3041..3043,3044..3046))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(2330..3040,3041..3043,3044..3046))
                     /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(2372..2393)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2721..2740
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2980..3001
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     misc_feature    3049..3074
                     /label=SA
                     /note="splice acceptor site"
     primer_bind     complement(3921..3940)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3922..3940)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3947..3964)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3947..3963)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3956..3978)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             4101..4403
                     /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     4282..4301
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             4752..5546
                     /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             4752..5546
                     /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(4806..4825)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5416..5435
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             complement(join(5796..6587,6588..6656))
                     /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     6419..6438
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6780..7368
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7269..7288
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7522..7539
                     /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 cctttacgag
      301 ccagtgatgg aagcaatgga aattaggcca agaaagggaa ggtccccggg tatccccctc
      361 ccacccttac ctcctgaaga ttttcattgt tgtcagcttc ctccacccac ttctgcagca
      421 agggccgcag cttacacatg ttcttgaagc taagctgcag agcctcaaag cggcagatgg
      481 tcgtttggct gaataccttc cctgggggag gccagtcaaa agagaagcaa aatgagggag
      541 cacgcagggc ccttgtgacc ctgagatcca agcttaccac ctcttcccag agggagctca
      601 aagcccaagc atcttctccc tctccctact cctcttcatg ggtgagggta gagtctgccc
      661 ctgcccctcc ccactaggtt cagggatact ccttagaggg gagatgcggt cagaatctgc
      721 agaggggaac ccaccaaata gaacccccag ggtgagcccc acatcggcct gtgtatatcc
      781 cagggtgatc ctcttctgct tcaggagctt ggcaaattgc tcgagttctt tctgcagagc
      841 tttgatgtcc tgggactgga ttttaaaagg cagaagactt gtaagaacat aaacacacca
      901 gttatcaatc tcccctttcc attcgggatt caagaaccta cgtgtggccc caaggaatag
      961 tctgtagaag tgcatctgcc ttccaagctg cccacctaac ttctagaaat aacctaccca
     1021 caaatgtcat tcacccattc cctgttcact gactcatggg atcctcgacc agcttctgat
     1081 ggaattagaa cttggcaaaa caatactgag aatgaagtgt atgtggaaca gaggctgctg
     1141 atctcgttct tcaggctatg aaactgacac atttggaaac cacagtactt agaaccacaa
     1201 agtgggaatc aagagaaaaa caatgatccc acgagagatc tatagatcta tagatcatga
     1261 gtgggaggaa tgagctggcc cttaatttgg ttttgcttgt ttaaattatg atatccaact
     1321 atgaaacatt atcataaagc aatagtaaag agccttcagt aaagagcagg catttatcta
     1381 atcccacccc acccccaccc ccgtagctcc aatccttcca ttcaaaatgt aggtactctg
     1441 ttctcaccct tcttaacaaa gtatgacagg aaaaacttcc attttagtgg acatctttat
     1501 tgtttaatag atcatcaatt tctgcatccc ggggatctga tatcatcgat gcatggggtc
     1561 gtgcgctcct ttcggtcggg cgctgcgggt cgtggggcgg gcgtcaggca ccgggcttgc
     1621 gggtcatgca ccaggtgcgc ggtccttcgg gcacctcgac gtcggcggtg acggtgaagc
     1681 cgagccgctc gtagaagggg aggttgcggg gcgcggaggt ctccaggaag gcgggcaccc
     1741 cggcgcgctc ggccgcctcc actccgggga gcacgacggc gctgcccaga cccttgccct
     1801 ggtggtcggg cgagacgccg acggtggcca ggaaccacgc gggctccttg ggccggtgcg
     1861 gcgccaggag gccttccatc tgttgctgcg cggccagccg ggaaccgctc aactcggcca
     1921 tgcgcgggcc gatctcggcg aacaccgccc ccgcttcgac gctctccggc gtggtccaga
     1981 ccgccaccgc ggcgccgtcg tccgcgaccc acaccttgcc gatgtcgagc ccgacgcgcg
     2041 tgaggaagag ttcttgcagc tcggtgaccc gctcgatgtg gcggtccgga tcgacggtgt
     2101 ggcgcgtggc ggggtagtcg gcgaacgcgg cggcgagggt gcgtacggcc ctggggacgt
     2161 cgtcgcgggt ggcgaggcgc accgtgggct tgtactcggt catggtaagc ttcagctgaa
     2221 gcttgcatgc gggcccgggg ttggactcga cgtctcccgc aagcttaaga aggtcaaaat
     2281 tcaaagtttg cttaccagaa ccgtcgactc tagagtcgcg accgctgtac ttgtacagct
     2341 cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc
     2401 gcttctcgtt ggggtctttg ctcagggcgg actgggtgct caggtagtgg ttgtcgggca
     2461 gcagcacggg gccgtcgccg atgggggtgt tctgctggta gtggtcggcg agctgcacgc
     2521 tgccgtcctc gatgttgtgg cggatcttga agttcacctt gatgccgttc ttctgcttgt
     2581 cggccatgat atagacgttg tggctgttgt agttgtactc cagcttgtgc cccaggatgt
     2641 tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg gttcaccagg gtgtcgccct
     2701 cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc cttgaagaag atggtgcgct
     2761 cctggacgta gccttcgggc atggcggact tgaagaagtc gtgctgcttc atgtggtcgg
     2821 ggtagcggct gaagcactgc acgccgtagg tcagggtggt cacgagggtg ggccagggca
     2881 cgggcagctt gccggtggtg cagatgaact tcagggtcag cttgccgtag gtggcatcgc
     2941 cctcgccctc gccggacacg ctgaacttgt ggccgtttac gtcgccgtcc agctcgacca
     3001 ggatgggcac caccccggtg aacagctcct cgcccttgct caccatggcc tgtgggagga
     3061 agagaagagg tcagggatcc catgtaacaa aggactactc ttcccccaga aactggcaca
     3121 tccaagggat gcagagcatc gtgaaaggac agaaagagag accctggcct cgaggagaac
     3181 acctgtcagg ttatgaaggt tagaagttct ttgctgggcg cggtggctca cgcctataat
     3241 tccagcactt tgggaggccg aggtgggcag atcacgaggt caggagttca agaccagcat
     3301 ggccaacatg gtgaaacccc gtctctacta aaaacacaaa aattagctgg gcacggtggc
     3361 acgcacctgt aatcccagct actcaggagg ctgaggcagg agaatcactt gaacccggga
     3421 ggcggaggtt gcagtgagct gagatcacgc cactgcactc cagcctgggt gacagagcaa
     3481 gactctgtct caaagaaaaa aaaaaagaag atagttcatt taatacctgc aaaattctct
     3541 cactcaagta tcacccccag tttaaggatg ttttgagatt agagaaatag ataagctgct
     3601 aagttctggg ttaattaaaa aggaagagca tcatgtctca gaagctaaat tcaatatata
     3661 ctctccccag cttgctttga gggtcccaca aactataaca tggcatgcat acacacaaac
     3721 acagcaaaaa agtaacaggt gtcataagaa tggataaagt gctttgtgtg tacttactcc
     3781 tcatttttta aattgattat ccctcatctt tactgtatct ttttcactat agaggcatcc
     3841 taattgattt ttaaattcaa gagatttatc gagcaccttc tataagcaag ggcgaattcg
     3901 cggccgctaa attcaattcg ccctatagtg agtcgtatta caattcactg gccgtcgttt
     3961 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     4021 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     4081 tgcgcagcct atacgtacgg cagtttaagg tttacaccta taaaagagag agccgttatc
     4141 gtctgtttgt ggatgtacag agtgatatta ttgacacgcc ggggcgacgg atggtgatcc
     4201 ccctggccag tgcacgtctg ctgtcagata aagtctcccg tgaactttac ccggtggtgc
     4261 atatcgggga tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg ccggtctccg
     4321 ttatcgggga agaagtggct gatctcagcc accgcgaaaa tgacatcaaa aacgccatta
     4381 acctgatgtt ctggggaata taaatgtcag gcatgagatt atcaaaaagg atcttcacct
     4441 agatcctttt cacgtagaaa gccagtccgc agaaacggtg ctgaccccgg atgaatgtca
     4501 gctactgggc tatctggaca agggaaaacg caagcgcaaa gagaaagcag gtagcttgca
     4561 gtgggcttac atggcgatag ctagactggg cggttttatg gacagcaagc gaaccggaat
     4621 tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac tggatggctt
     4681 tcttgccgcc aaggatctga tggcgcaggg gatcaagctc tgatcaagag acaggatgag
     4741 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     4801 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     4861 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     4921 tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     4981 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     5041 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     5101 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     5161 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     5221 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga
     5281 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     5341 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     5401 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     5461 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     5521 atcgccttct tgacgagttc ttctgaatta ttaacgctta caatttcctg atgcggtatt
     5581 ttctccttac gcatctgtgc ggtatttcac accgcatcag gtggcacttt tcggggaaat
     5641 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     5701 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5761 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5821 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5881 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5941 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     6001 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     6061 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     6121 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     6181 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     6241 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     6301 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     6361 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     6421 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6481 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     6541 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6601 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6661 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgac caaaatccct
     6721 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6781 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6841 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6901 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     6961 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     7021 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     7081 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     7141 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     7201 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     7261 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     7321 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     7381 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     7441 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     7501 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaag
//
