LOCUS       sequence-54252-e        7484 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 7484)
  AUTHORS   Dubarry M, Loiodice I, Chen CL, Thermes C, Taddei A
  TITLE     Tight protein-DNA interactions favor gene silencing.
  JOURNAL   Genes Dev. 2011 Jul 1;25(13):1365-70.
  PUBMED    21724830
REFERENCE   2  (bases 1 to 7484)
  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: "Genes 
            Dev."; date: "2011-07-1"; volume: "25(13)"; pages: "1365-70"
COMMENT     Sequence Label: pAT222
FEATURES             Location/Qualifiers
     source          1..7484
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      725..1313
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1214..1233
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1467..1484
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(1637..1642,1643..1660,1661..1667)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1675..1691
                     /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     1680..1702
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1699..1715
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1699..1715
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1736..1754
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1792..1979
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             1989..2702
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2019..2047)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2642..2662
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(2749..2768)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3798..3818
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3862..3881)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3863..3881)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3888..3905)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3888..3904)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3897..3919)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4049..4504
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4136..4155)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4346..4367
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4804..5208
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             5221..6315
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVAGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANLLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(6808..6827)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6927..6949
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6987..7005)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7073..7177
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7178..7246,7247..7484,1..554)
                     /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     complement(7396..7415)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
       61 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
      121 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
      181 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
      241 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
      301 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
      361 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
      421 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
      481 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
      541 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
      601 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
      661 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
      721 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
      781 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
      841 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
      901 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
      961 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     1021 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     1081 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     1141 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     1201 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     1261 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     1321 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     1381 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     1441 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     1501 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     1561 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tacctcactc
     1621 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     1681 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctcggaatta
     1741 accctcacta aagggaacaa aagctgggta ccctattact cttggcctcc tctagtacac
     1801 tctatatttt tttatgcctc ggtaatgatt ttcatttttt tttttccacc tagcggatga
     1861 ctcttttttt ttcttagcga ttggcattat cacataatga attatacatt atataaagta
     1921 atgtgatttc ttcgaagaat atactaaaaa atgagcaggc aagataaacg aaggcaaagc
     1981 tcgaggagat gagtaaagga gaagaacttt tcactggagt tgtcccaatt cttgttgaat
     2041 tagatggtga tgttaatggg cacaaatttt ctgtcagtgg agagggtgaa ggtgatgcaa
     2101 catacggaaa acttaccctt aaatttattt gcactactgg aaaactacct gttccatggc
     2161 caacacttgt cactactttc acttatggtg ttcaatgctt ttcaagatac ccagatcata
     2221 tgaaacagca tgactttttc aagagtgcca tgcccgaagg ttatgtacag gaaagaacta
     2281 tatttttcaa agatgacggg aactacaaga cacgtgctga agtcaagttt gaaggtgata
     2341 cccttgttaa tagaatcgag ttaaaaggta ttgattttaa agaagatgga aacattcttg
     2401 gacacaaatt ggaatacaac tataactcac acaatgtata catcatggca gacaaacaaa
     2461 agaatggaat caaagctaac ttcaaaatta gacacaacat tgaagatgga agcgttcaac
     2521 tagcagacca ttatcaacaa aatactccaa ttggcgatgg ccctgtcctt ttaccagaca
     2581 accattacct gtccacacaa tctgcccttt cgaaagatcc caacgaaaag agagaccaca
     2641 tggtccttct tgagtttgta acagctgctg ggattacaca tggcatggat gaactataca
     2701 aagaattcct gcagcccggg ggatccatgg tgaaatatgt aacgttatac gatgtcgcag
     2761 agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt
     2821 ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc
     2881 gcgtggcaca gcaactggcg gacaaaggtg gcggatcgtt gctgattggc gttgcctcct
     2941 ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc
     3001 aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag
     3061 cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg
     3121 atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg
     3181 atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac
     3241 tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat
     3301 taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc
     3361 aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa
     3421 ccatgctaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga
     3481 tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct
     3541 cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca
     3601 tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc
     3661 agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca
     3721 ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     3781 tgggatcctg tactccacca aagaagaaga gaaaggttgc ctaatctaga gcggccgcca
     3841 ccgcggtgga gctccaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt
     3901 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     3961 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     4021 ttgcgcagcc tgaatggcga atggcgcgac gcgccctgta gcggcgcatt aagcgcggcg
     4081 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     4141 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4201 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4261 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     4321 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     4381 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     4441 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     4501 atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatcga
     4561 cggtcgagga gaacttctag tatatccaca tacctaatat tattgcctta ttaaaaatgg
     4621 aatcccaaca attacatcaa aatccacatt ctcttcaaaa tcaattgtcc tgtacttcct
     4681 tgttcatgtg tgttcaaaaa cgttatattt ataggataat tatactctat ttctcaacaa
     4741 gtaattggtt gtttggccga gcggtctaag gcgcctgatt caagaaatat cttgaccgca
     4801 gttaactgtg ggaatactca ggtatcgtaa gatgcaagag ttcgaatctc ttagcaacca
     4861 ttattttttt cctcaacata acgagaacac acaggggcgc tatcgcacag aatcaaattc
     4921 gatgactgga aattttttgt taatttcaga ggtcgcctga cgcatatacc tttttcaact
     4981 gaaaaattgg gagaaaaagg aaaggtgaga ggccggaacc ggcttttcat atagaataga
     5041 gaagcgttca tgactaaatg cttgcatcac aatacttgaa gttgacaata ttatttaagg
     5101 acctattgtt ttttccaata ggtggttagc aatcgtctta ctttctaact tttcttacct
     5161 tttacatttc agcaatatat atatatattt caaggatata ccattctaat gtctgcccct
     5221 atgtctgccc ctaagaagat cgtcgttttg ccaggtgacc acgttggtca agaaatcaca
     5281 gccgaagcca ttaaggttct taaagctatt tctgatgttc gttccaatgt caagttcgat
     5341 ttcgaaaatc atttaattgg tggtgctgct atcgatgcta caggtgtccc acttccagat
     5401 gaggcgctgg aagcctccaa gaaggttgat gccgttttgt taggtgctgt ggctggtcct
     5461 aaatggggta ccggtagtgt tagacctgaa caaggtttac taaaaatccg taaagaactt
     5521 caattgtacg ccaacttaag accatgtaac tttgcatccg actctctttt agacttatct
     5581 ccaatcaagc cacaatttgc taaaggtact gacttcgttg ttgtcagaga attagtggga
     5641 ggtatttact ttggtaagag aaaggaagac gatggtgatg gtgtcgcttg ggatagtgaa
     5701 caatacaccg ttccagaagt gcaaagaatc acaagaatgg ccgctttcat ggccctacaa
     5761 catgagccac cattgcctat ttggtccttg gataaagcta atcttttggc ctcttcaaga
     5821 ttatggagaa aaactgtgga ggaaaccatc aagaacgaat tccctacatt gaaggttcaa
     5881 catcaattga ttgattctgc cgccatgatc ctagttaaga acccaaccca cctaaatggt
     5941 attataatca ccagcaacat gtttggtgat atcatctccg atgaagcctc cgttatccca
     6001 ggttccttgg gtttgttgcc atctgcgtcc ttggcctctt tgccagacaa gaacaccgca
     6061 tttggtttgt acgaaccatg ccacggttct gctccagatt tgccaaagaa taaggttgac
     6121 cctatcgcca ctatcttgtc tgctgcaatg atgttgaaat tgtcattgaa cttgcctgaa
     6181 gaaggtaagg ccattgaaga tgcagttaaa aaggttttgg atgcaggtat cagaactggt
     6241 gatttaggtg gttccaacag taccaccgaa gtcggtgatg ctgtcgccga agaagttaag
     6301 aaaatccttg cttaaaaaga ttctcttttt ttatgatatt tgtacataaa ctttataaat
     6361 gaaattcata atagaaacga cacgaaatta caaaatggaa tatgttcata gggtagacga
     6421 aactatatac gcaatctaca tacatttatc aagaaggaga aaaaggagga tagtaaagga
     6481 atacaggtaa gcaaattgat actaatggct caacgtgata aggaaaaaga attgcacttt
     6541 aacattaata ttgacaagga ggagggcacc acacaaaaag ttaggtgtaa cagaaaatca
     6601 tgaaactacg attcctaatt tgatattgga ggattttctc taaaaaaaaa aaaatacaac
     6661 aaataaaaaa cactcaatga cctgaccatt tgatggagtt taagtcaata ccttcttgaa
     6721 gcatttccca taatggtgaa agttccctca agaattttac tctgtcagaa acggccttac
     6781 gacgtagtcg atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     6841 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     6901 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     6961 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     7021 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     7081 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     7141 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     7201 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     7261 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     7321 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     7381 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     7441 actcggtcgc cgcatacact attctcagaa tgacttggtt gagt
//
