LOCUS       sequence-59474-e        5772 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 5772)
  AUTHORS   Crook NC, Freeman ES, Alper HS
  TITLE     Re-engineering multicloning sites for function and convenience.
  JOURNAL   Nucleic Acids Res. 2011 Aug;39(14):e92. doi: 10.1093/nar/gkr346. 
            Epub 2011 May 17.
  PUBMED    21586584
REFERENCE   2  (bases 1 to 5772)
  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: "Nucleic 
            Acids Res."; date: "2011-08"; volume: "39(14):e92. doi"; pages: " 
            10.1093/nar/gkr346. Epub 2011 May 17"
COMMENT     Sequence Label: pGPD2
FEATURES             Location/Qualifiers
     source          1..5772
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    70..573
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        610..714
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(715..783,784..1575)
                     /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(933..952)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1746..2334
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2235..2254
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2488..2505
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(2658..2663,2664..2681,2682..2688)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2696..2712
                     /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     2701..2723
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2720..2736
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2720..2736
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2755..2775
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2757..2775
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2800..3443
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     3337..3360
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     complement(3528..3546)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     terminator      3558..3747
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3777..3796)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3778..3796)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3806..3823)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3806..3822)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3815..3837)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3967..4422
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4054..4073)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4264..4285
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4553..5356)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(5357..5577)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5603..5622)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5722..5744
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttaggacgg atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa tgatggaata
      121 atttgggaat ttactctgtg tttatttatt tttatgtttt gtatttggat tttagaaagt
      181 aaataaagaa ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa
      241 atttcaacaa aaagcgtact ttacatatat atttattaga caagaaaagc agattaaata
      301 gatatacatt cgattaacga taagtaaaat gtaaaatcac aggattttcg tgtgtggtct
      361 tctacacaga caagatgaaa caattcggca ttaatacctg agagcaggaa gagcaagata
      421 aaaggtagta tttgttggcg atccccctag agtcttttac atcttcggaa aacaaaaact
      481 attttttctt taatttcttt ttttactttc tatttttaat ttatatattt atattaaaaa
      541 atttaaatta taattatttt tatagcacgt gatgaaaagg acccaggtgg cacttttcgg
      601 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
      661 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
      721 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
      781 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
      841 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
      901 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
      961 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     1021 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     1081 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     1141 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     1201 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     1261 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     1321 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     1381 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     1441 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     1501 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     1561 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     1621 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     1681 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1741 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1801 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1861 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     1921 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     1981 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     2041 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     2101 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     2161 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     2221 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     2281 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     2341 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     2401 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     2461 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     2521 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     2581 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttacctcact
     2641 cattaggcac cccaggcttt acactttatg cttccggctc ctatgttgtg tggaattgtg
     2701 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt
     2761 aaccctcact aaagggaaca aaagctggag ctcagtttat cattatcaat actcgccatt
     2821 tcaaagaata cgtaaataat taatagtagt gattttccta actttattta gtcaaaaaat
     2881 tagcctttta attctgctgt aacccgtaca tgcccaaaat agggggcggg ttacacagaa
     2941 tatataacat cgtaggtgtc tgggtgaaca gtttattcct ggcatccact aaatataatg
     3001 gagcccgctt tttaagctgg catccagaaa aaaaaagaat cccagcacca aaatattgtt
     3061 ttcttcacca accatcagtt cataggtcca ttctcttagc gcaactacag agaacagggg
     3121 cacaaacagg caaaaaacgg gcacaacctc aatggagtga tgcaacctgc ctggagtaaa
     3181 tgatgacaca aggcaattga cccacgcatg tatctatctc attttcttac accttctatt
     3241 accttctgct ctctctgatt tggaaaaagc tgaaaaaaaa ggttgaaacc agttccctga
     3301 aattattccc ctacttgact aataagtata taaagacggt aggtattgat tgtaattctg
     3361 taaatctatt tcttaaactt cttaaattct acttttatag ttagtctttt ttttagtttt
     3421 aaaacaccag aacttagttt cgacggataa agcttactag tatcgataaa gaattctcta
     3481 gaggatccct gcagctcgag gtcgaccccg gggatatcat tagttatgtc acgcttacat
     3541 tcacgccctc cccccacatc cgctctaacc gaaaaggaag gagttagaca acctgaagtc
     3601 taggtcccta tttatttttt tatagttatg ttagtattaa gaacgttatt tatatttcaa
     3661 atttttcttt tttttctgta cagacgcgtg tacgcatgta acattatact gaaaaccttg
     3721 cttgagaagg ttttgggacg ctcgaaggct ttaatttgcg gccggtaccc aattcgccct
     3781 atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     3841 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     3901 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     3961 ggcgcgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     4021 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     4081 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     4141 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     4201 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4261 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     4321 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     4381 aatttaacgc gaattttaac aaaatattaa cgtttacaat ttcctgatgc ggtattttct
     4441 ccttacgcat ctgtgcggta tttcacaccg catagggtaa taactgatat aattaaattg
     4501 aagctctaat ttgtgagttt agtatacatg catttactta taatacagtt ttttagtttt
     4561 gctggccgca tcttctcaaa tatgcttccc agcctgcttt tctgtaacgt tcaccctcta
     4621 ccttagcatc ccttcccttt gcaaatagtc ctcttccaac aataataatg tcagatcctg
     4681 tagagaccac atcatccacg gttctatact gttgacccaa tgcgtctccc ttgtcatcta
     4741 aacccacacc gggtgtcata atcaaccaat cgtaaccttc atctcttcca cccatgtctc
     4801 tttgagcaat aaagccgata acaaaatctt tgtcgctctt cgcaatgtca acagtaccct
     4861 tagtatattc tccagtagat agggagccct tgcatgacaa ttctgctaac atcaaaaggc
     4921 ctctaggttc ctttgttact tcttctgccg cctgcttcaa accgctaaca atacctgggc
     4981 ccaccacacc gtgtgcattc gtaatgtctg cccattctgc tattctgtat acacccgcag
     5041 agtactgcaa tttgactgta ttaccaatgt cagcaaattt tctgtcttcg aagagtaaaa
     5101 aattgtactt ggcggataat gcctttagcg gcttaactgt gccctccatg gaaaaatcag
     5161 tcaagatatc cacatgtgtt tttagtaaac aaattttggg acctaatgct tcaactaact
     5221 ccagtaattc cttggtggta cgaacatcca atgaagcaca caagtttgtt tgcttttcgt
     5281 gcatgatatt aaatagcttg gcagcaacag gactaggatg agtagcagca cgttccttat
     5341 atgtagcttt cgacatgatt tatcttcgtt tcctgcaggt ttttgttctg tgcagttggg
     5401 ttaagaatac tgggcaattt catgtttctt caacactaca tatgcgtata tataccaatc
     5461 taagtctgtg ctccttcctt cgttcttcct tctgttcgga gattaccgaa tcaaaaaaat
     5521 ttcaaagaaa ccgaaatcaa aaaaaagaat aaaaaaaaaa tgatgaattg aattgaaaag
     5581 ctgtggtatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     5641 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     5701 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     5761 cgaaacgcgc ga
//
