LOCUS       sequence-46773-e        5498 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 5498)
  AUTHORS   Bhakta MS, Segal DJ
  TITLE     The generation of zinc finger proteins by modular assembly.
  JOURNAL   Methods Mol Biol. 2010;649:3-30.
  PUBMED    20680825
REFERENCE   2  (bases 1 to 5498)
  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"; journalName: "Methods Mol
            Biol."; date: "2010"; volume: "649"; pages: "3-30"
COMMENT     Sequence Label: pPGK-GZF3-DD
FEATURES             Location/Qualifiers
     source          1..5498
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        15..515
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     434..452
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     promoter        664..682
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     664..681
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     695..714
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             761..787
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             815..835
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1121..1705
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="LVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVM
                     EFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQDY
                     VEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLS
                     VEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     regulatory      1742..1751
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1755..1889
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1792..1811)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1846..1865
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1962..2319
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1962..2153
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1985..2005)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2170..2305
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2339..2356)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2339..2355)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2348..2370)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2568..3023
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2655..2674)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2865..2886
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3040..3059)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3194..3216
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3269..3287)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3355..3459
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3645..4023
                     /label=ISS
                     /note="immunostimulatory sequence from the AmpR gene; 
                     contains unmethylated CpG dinucleotides in the context of 
                     5'-AACGTT-3' (Sato et al., 1996)"
     primer_bind     complement(3679..3698)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4492..5080
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4981..5000
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5234..5251
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(5404..5409,5410..5427,5428..5434)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5442..5458
                     /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     5447..5469
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5466..5482
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5466..5482
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 tcgagctctt ggggttgcgc cttttccaag gcagccctgg gtttgcgcag ggacgcggct
       61 gctctgggcg tggttccggg aaacgcagcg gcgccgaccc tgggtctcgc acattcttca
      121 cgtccgttcg cagcgtcacc cggatcttcg ccgctaccct tgtgggcccc ccggcgacgc
      181 ttcctgctcc gcccctaagt cgggaaggtt ccttgcggtt cgcggcgtgc cggacgtgac
      241 aaacggaagc cgcacgtctc actagtaccc tcgcagacgg acagcgccag ggagcaatgg
      301 cagcgcgccg accgcgatgg gctgtggcca atagcggctg ctcagcgggg cgcgccgaga
      361 gcagcggccg ggaaggggcg gtgcgggagg cggggtgtgg ggcggtagtg tgggccctgt
      421 tcctgcccgc gcggtgttcc gcattctgca agcctccgga gcgcacgtcg gcagtcggct
      481 ccctcgttga ccgaatcacc gacctctctc cccagggccg cggccgggaa cggtgcattg
      541 gaacgcggat tccccgtgcc aagagtgacg taagtaccgc ctatagagtc tatggcccac
      601 ccccttggct tcgttagaac gcggctacaa ttaatacata accttatgta tcatacacat
      661 acgatttagg tgacactata gaataacatc cactttgcct ttctctccac aggtgtccac
      721 tcccaggtcc aactgcacct cggttctatc gattgacatg tacccatacg atgtcccaga
      781 ctacgcgaat tccccgggga tcccaggcat ggggcccaaa aagaaacgca aagttgggcg
      841 cctcgagccc ggggagaagc cctatgcttg tccggaatgt ggtaagtcct tcagcgacag
      901 cgggaacctg cgcgtccacc agcgtaccca cacgggtgaa aaaccgtata aatgcccaga
      961 gtgcggcaaa tcttttagca ccagcggcaa cctggtgcgc catcaacgca ctcatactgg
     1021 cgagaagcca tacaaatgtc cagaatgtgg caagtctttc tcccgcagcg atcacctggc
     1081 cgaacaccaa cgtactcaca ccggtgcggc cgcccgcgcc ctagtcaaaa gtgaactgga
     1141 ggagaagaaa tctgaacttc gtcataaatt gaaatatgtg cctcatgaat atattgaatt
     1201 aattgaaatt gccagaaatt ccactcagga tagaattctt gaaatgaagg taatggaatt
     1261 ttttatgaaa gtttatggat atagaggtaa acatttgggt ggatcaagga aaccggacgg
     1321 agcaatttat actgtcggat ctcctattga ttacggtgtg atcgtggata ctaaagctta
     1381 tagcggaggt tataatctgc caattggcca agcagatgaa atgcaagatt atgtcgaaga
     1441 aaatcaaaca cgaaacaaac atatcaaccc taatgaatgg tggaaagtct atccatcttc
     1501 tgtaacggaa tttaagtttt tatttgtgag tggtcacttt aaaggaaact acaaagctca
     1561 gcttacacga ttaaatcata tcactaattg taatggagct gttcttagtg tagaagagct
     1621 tttaattggt ggagaaatga ttaaagccgg cacattaacc ttagaggaag tgagacggaa
     1681 atttaataac ggcgagataa acttttaatt aactaatcta gagtcgacct gcagaagctt
     1741 ggccgccatg gcccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     1801 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     1861 tcatcaatgt atcttatcat gtctggatcg atcgggaatt aattcggcgc agcaccatgg
     1921 cctgaaataa cctctgaaag aggaacttgg ttaggtacct tctgaggcgg aaagaaccag
     1981 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     2041 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     2101 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     2161 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     2221 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     2281 tagtgaggag gcttttttgg aggcctaggc atttgcaaaa agctgttaac agcttggcac
     2341 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     2401 ttgcagcaca tccccccttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     2461 cttcccaaca gttgcgtagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta
     2521 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
     2581 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2641 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2701 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2761 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
     2821 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2881 aactggaaca acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc
     2941 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     3001 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
     3061 atagttaagc caactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     3121 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     3181 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     3241 gcgaggcagt attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     3301 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     3361 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3421 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     3481 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     3541 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     3601 gatctcaaca gcggtaagat cctttgagag ttttcgcccc gaagaacgtt ttccaatgat
     3661 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgatgacg ccgggcaaga
     3721 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     3781 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     3841 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     3901 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     3961 gaatgaagcc ataccaaacg acgagcgtga caccacgatg ccagcagcaa tggcaacaac
     4021 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     4081 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     4141 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     4201 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     4261 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     4321 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     4381 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     4441 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttcagatcc
     4501 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     4561 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     4621 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4681 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4741 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4801 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4861 actgagatac ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc
     4921 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     4981 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     5041 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     5101 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     5161 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     5221 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     5281 gcctctcccc gcgcgttggc cgattcatta atccagctgg cacgacaggt ttcccgactg
     5341 gaaagcgggc agtgagcgca acgcaattaa tgtgagttac ctcactcatt aggcacccca
     5401 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     5461 tcacacagga aacagctatg accatgatta cgaattaa
//
