LOCUS       sequence-59473-e        5548 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 5548)
  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 5548)
  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: pTEF2
FEATURES             Location/Qualifiers
     source          1..5548
                     /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        2793..3191
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     complement(3304..3322)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     terminator      3334..3523
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3553..3572)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3554..3572)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3582..3599)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3582..3598)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3591..3613)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3743..4198
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3830..3849)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4040..4061
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4329..5132)
                     /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(5133..5353)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5379..5398)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5498..5520
                     /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 ctcatagctt caaaatgttt ctactccttt
     2821 tttactcttc cagattttct cggactccgc gcatcgccgt accacttcaa aacacccaag
     2881 cacagcatac taaatttccc ctctttcttc ctctagggtg tcgttaatta cccgtactaa
     2941 aggtttggaa aagaaaaaag agaccgcctc gtttcttttt cttcgtcgaa aaaggcaata
     3001 aaaattttta tcacgtttct ttttcttgaa aatttttttt ttgatttttt tctctttcga
     3061 tgacctccca ttgatattta agttaataaa cggtcttcaa tttctcaagt ttcagtttca
     3121 tttttcttgt tctattacaa ctttttttac ttcttgctca ttagaaagaa agcatagcaa
     3181 tctaatctaa gtttacccca tcgatcaccc actagttcta gacccccgaa ttccccccaa
     3241 gctttaaagt cgaccccccc tcgagacccc ggatcccccg ggctgcagga tatcattagt
     3301 tatgtcacgc ttacattcac gccctccccc cacatccgct ctaaccgaaa aggaaggagt
     3361 tagacaacct gaagtctagg tccctattta tttttttata gttatgttag tattaagaac
     3421 gttatttata tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg catgtaacat
     3481 tatactgaaa accttgcttg agaaggtttt gggacgctcg aaggctttaa tttgcggccg
     3541 gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
     3601 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     3661 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     3721 agcctgaatg gcgaatggcg cgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3781 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     3841 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3901 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     3961 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     4021 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     4081 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     4141 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttcc
     4201 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata gggtaataac
     4261 tgatataatt aaattgaagc tctaatttgt gagtttagta tacatgcatt tacttataat
     4321 acagtttttt agttttgctg gccgcatctt ctcaaatatg cttcccagcc tgcttttctg
     4381 taacgttcac cctctacctt agcatccctt ccctttgcaa atagtcctct tccaacaata
     4441 ataatgtcag atcctgtaga gaccacatca tccacggttc tatactgttg acccaatgcg
     4501 tctcccttgt catctaaacc cacaccgggt gtcataatca accaatcgta accttcatct
     4561 cttccaccca tgtctctttg agcaataaag ccgataacaa aatctttgtc gctcttcgca
     4621 atgtcaacag tacccttagt atattctcca gtagataggg agcccttgca tgacaattct
     4681 gctaacatca aaaggcctct aggttccttt gttacttctt ctgccgcctg cttcaaaccg
     4741 ctaacaatac ctgggcccac cacaccgtgt gcattcgtaa tgtctgccca ttctgctatt
     4801 ctgtatacac ccgcagagta ctgcaatttg actgtattac caatgtcagc aaattttctg
     4861 tcttcgaaga gtaaaaaatt gtacttggcg gataatgcct ttagcggctt aactgtgccc
     4921 tccatggaaa aatcagtcaa gatatccaca tgtgttttta gtaaacaaat tttgggacct
     4981 aatgcttcaa ctaactccag taattccttg gtggtacgaa catccaatga agcacacaag
     5041 tttgtttgct tttcgtgcat gatattaaat agcttggcag caacaggact aggatgagta
     5101 gcagcacgtt ccttatatgt agctttcgac atgatttatc ttcgtttcct gcaggttttt
     5161 gttctgtgca gttgggttaa gaatactggg caatttcatg tttcttcaac actacatatg
     5221 cgtatatata ccaatctaag tctgtgctcc ttccttcgtt cttccttctg ttcggagatt
     5281 accgaatcaa aaaaatttca aagaaaccga aatcaaaaaa aagaataaaa aaaaaatgat
     5341 gaattgaatt gaaaagctgt ggtatggtgc actctcagta caatctgctc tgatgccgca
     5401 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     5461 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     5521 ttttcaccgt catcaccgaa acgcgcga
//
