LOCUS       sequence-54256-e        6433 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 6433)
  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 6433)
  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: pAT253
FEATURES             Location/Qualifiers
     source          1..6433
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /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(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1721..1740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1974..1991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(2144..2149,2150..2167,2168..2174)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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     2187..2209
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2206..2222
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2243..2261
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2299..2486
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             2496..3209
                     /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(2526..2554)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3149..3169
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(3256..3275)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4305..4325
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4369..4388)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4370..4388)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4395..4412)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4395..4411)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4404..4426)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4556..5011
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4643..4662)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4853..4874
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5271..5930)
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALLARGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHFL
                     ESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     promoter        complement(5931..6117)
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     primer_bind     complement(6264..6283)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6383..6405
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttac ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     2221 accatgatta cgccaagctc ggaattaacc ctcactaaag ggaacaaaag ctgggtaccc
     2281 tattactctt ggcctcctct agtacactct atattttttt atgcctcggt aatgattttc
     2341 attttttttt ttccacctag cggatgactc tttttttttc ttagcgattg gcattatcac
     2401 ataatgaatt atacattata taaagtaatg tgatttcttc gaagaatata ctaaaaaatg
     2461 agcaggcaag ataaacgaag gcaaagctcg aggagatgag taaaggagaa gaacttttca
     2521 ctggagttgt cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg
     2581 tcagtggaga gggtgaaggt gatgcaacat acggaaaact tacccttaaa tttatttgca
     2641 ctactggaaa actacctgtt ccatggccaa cacttgtcac tactttcact tatggtgttc
     2701 aatgcttttc aagataccca gatcatatga aacagcatga ctttttcaag agtgccatgc
     2761 ccgaaggtta tgtacaggaa agaactatat ttttcaaaga tgacgggaac tacaagacac
     2821 gtgctgaagt caagtttgaa ggtgataccc ttgttaatag aatcgagtta aaaggtattg
     2881 attttaaaga agatggaaac attcttggac acaaattgga atacaactat aactcacaca
     2941 atgtatacat catggcagac aaacaaaaga atggaatcaa agctaacttc aaaattagac
     3001 acaacattga agatggaagc gttcaactag cagaccatta tcaacaaaat actccaattg
     3061 gcgatggccc tgtcctttta ccagacaacc attacctgtc cacacaatct gccctttcga
     3121 aagatcccaa cgaaaagaga gaccacatgg tccttcttga gtttgtaaca gctgctggga
     3181 ttacacatgg catggatgaa ctatacaaag aattcctgca gcccggggga tccatggtga
     3241 aatatgtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     3301 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     3361 tggcggagct gaattacatt cccaaccgcg tggcacagca actggcggac aaaggtggcg
     3421 gatcgttgct gattggcgtt gcctcctcca gtctggccct gcacgcgccg tcgcaaattg
     3481 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
     3541 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
     3601 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
     3661 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
     3721 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
     3781 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
     3841 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
     3901 ggagtgccat gtccggtttt caacaaacca tgctaatgct gaatgagggc atcgttccca
     3961 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
     4021 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct
     4081 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     4141 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     4201 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     4261 gcgcgttggc cgattcatta atgcagctgg gatcctgtac tccaccaaag aagaagagaa
     4321 aggttgccta atctagagcg gccgccaccg cggtggagct ccaattcgcc ctatagtgag
     4381 tcgtattaca attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     4441 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     4501 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcgacgcg
     4561 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     4621 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     4681 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     4741 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     4801 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4861 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     4921 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     4981 aattttaaca aaatattaac gtttacaatt tcctgatgcg gtattttctc cttacgcatc
     5041 tgtgcggtat ttcacaccgc atagatccgt cgagttcaag agaaaaaaaa agaaaaagca
     5101 aaaagaaaaa aggaaagcgc gcctcgttca gaatgacacg tatagaatga tgcattacct
     5161 tgtcatcttc agtatcatac tgttcgtata catacttact gacattcata ggtatacata
     5221 tatacacatg tatatatatc gtatgctgca gctttaaata atcggtgtca ctacataaga
     5281 acacctttgg tggagggaac atcgttggta ccattgggcg aggtggcttc tcttatggca
     5341 accgcaagag ccttgaacgc actctcacta cggtgatgat cattcttgcc tcgcagacaa
     5401 tcaacgtgga gggtaattct gctagcctct gcaaagcttt caagaaaatg cgggatcatc
     5461 tcgcaagaga gatctcctac tttctccctt tgcaaaccaa gttcgacaac tgcgtacggc
     5521 ctgttcgaaa gatctaccac cgctctggaa agtgcctcat ccaaaggcgc aaatcctgat
     5581 ccaaaccttt ttactccacg cgccagtagg gcctctttaa aagcttgacc gagagcaatc
     5641 ccgcagtctt cagtggtgtg atggtcgtct atgtgtaagt caccaatgca ctcaacgatt
     5701 agcgaccagc cggaatgctt ggccagagca tgtatcatat ggtccagaaa ccctatacct
     5761 gtgtggacgt taatcacttg cgattgtgtg gcctgttctg ctactgcttc tgcctctttt
     5821 tctgggaaga tcgagtgctc tatcgctagg ggaccaccct ttaaagagat cgcaatctga
     5881 atcttggttt catttgtaat acgctttact agggctttct gctctgtcat ctttgccttc
     5941 gtttatcttg cctgctcatt ttttagtata ttcttcgaag aaatcacatt actttatata
     6001 atgtataatt cattatgtga taatgccaat cgctaagaaa aaaaaagagt catccgctag
     6061 gggaaaaaaa aaaatgaaaa tcattaccga ggcataaaaa aatatagagt gtactagagg
     6121 aggccaagag taatagaaaa agaaaattgc gggaaaggac tgtgttatga cttccctgac
     6181 taatgccgtg ttcaaacgat acctggcagt gactcctagc gctcaccaag ctcttaaaac
     6241 gggaatttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     6301 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     6361 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     6421 ccgaaacgcg cga
//
