LOCUS       sequence-12877-e        5903 bp    DNA     circular SYN 15-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5903)
  AUTHORS   Cross FR, Pecani K
  TITLE     Efficient and rapid exact gene replacement without selection.
  JOURNAL   Yeast. 2010 Oct 22. ():.
  PUBMED    20973105
REFERENCE   2  (bases 1 to 5903)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Yeast. 2010
            Oct 22. ():."
COMMENT     Sequence Label: FC510
FEATURES             Location/Qualifiers
     source          1..5903
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        196..416
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             417..1220
                     /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"
     rep_origin      complement(1351..1806)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1488..1509)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1700..1719
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1936..1958
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1950..1967
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1951..1967
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1977..1996
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1977..1995
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     2020..2036
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2021..2037
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2071..2087)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2071..2087)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3643..3663)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3643..3661)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3682..3698)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3682..3698)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3695..3717)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3706..3722
                     /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)."
     promoter        complement(join(3730..3736,3737..3754,3755..3760))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3913..3930)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4084..4672)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4164..4183)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4843..5634,5635..5703))
                     /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     5466..5485
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5704..5808)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5876..5894
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc
      241 ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg
      301 agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc
      361 cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt
      421 cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat
      481 ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca
      541 aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg
      601 tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg
      661 ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca
      721 aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac
      781 acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa
      841 aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg
      901 gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct
      961 ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac
     1021 ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg
     1081 atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa
     1141 gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa
     1201 gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac
     1261 aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac
     1321 agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat
     1381 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     1441 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     1501 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     1561 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     1621 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     1681 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     1741 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     1801 gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     1861 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     1921 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat
     1981 acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac ggtatcgata
     2041 agcttgatat cgaattcctg cagcccgggg gatccactag ttctagatct cgagatagtt
     2101 tttatacaat acatatatat atatatatat atatatttac aagaaaagca tggcttatgt
     2161 attatacttg cttatgtacc aatatataca tatatggcct tattagggca acaaacgcca
     2221 aaaataaaaa cgaacctaaa tgagaaaaaa agagttaaca aaaatttgga aaacaacaag
     2281 aactcaaaaa aaaaaattgg tggaaggacc aagtcctctt gaaagaaaat ttaatactgc
     2341 ttttccagaa tatcatattg gaaaaaatat tcaagccagc acatcagcta cagtggaaaa
     2401 tagcccagat caaatagaac tatccttcgc atatgagcgg tgaattagca aattacaaaa
     2461 gacttgagaa agtcggtgaa ggtacatacg gtgttgttta taaagcgtta gacttaagac
     2521 ctggccaagg tcaaagagta gtcgcattga agaaaataag actagagagt gaagacgagg
     2581 gtgttcccag tacagccatc agagaaatct cattattgaa ggaattaaaa gacgataata
     2641 ttgtcagatt atacgatatt gttcactctg atgcacacaa gctatatctt gtttttgagt
     2701 tcctcgattt ggacctgaaa agatatatgg agggtattcc aaaggaccaa ccgttaggag
     2761 ctgatattgt taagaagttt atgatgcaac tttgtaaggg tattgcatac tgccactcac
     2821 accgtattct gcatcgtgat ttaaaaccgc agaacttatt gattaacaaa gatgggaatc
     2881 taaaactagg tgattttggc ttagcgcgtg cttttggtgt tccgttgaga gcttacacac
     2941 atgaaattgt tactctatgg tatagagctc cggaggtatt actgggtgga aaacaatata
     3001 gtacaggtgt cgatacatgg tccatcggct gtatatttgc cgaaatgtgt aacaggaaac
     3061 caatcttcag tggcgatagt gagatcgatc agattttcaa gatattcaga gtattgggaa
     3121 cgccgaatga agctatatgg ccagatattg tctacttgcc tgatttcaag ccaagctttc
     3181 ctcaatggcg cagaaaagac ctatcacaag tggtaccaag tctagatcca cgcggtattg
     3241 atttgttgga caaactcctc gcgtatgacc ctattaaccg gattagcgcc agaagagcag
     3301 ccatccaccc ctacttccaa gaatcataag cattataatc tatttcgcta ttatattaca
     3361 aatgctactg cactgtcatt atagcctagt aaagtatata gtgaatacaa tatactcagt
     3421 ttcaacatta tgatgggtaa ctccatcaga aaatatattc atcgtcatat acggaacatt
     3481 cagttaagat ctaaataaat tcgttttcaa tgattaaaat agcatagtcg ggtttttctt
     3541 ttagtttcag ctttccgcaa cagtataatt ttataaacct ggttttggtt ttgtagagtt
     3601 gttgacgcgg ccgccaccgc ggtggagctc cagcttttgt tccctttagt gagggttaat
     3661 tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     3721 aattccacac aacataggag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     3781 gaggtaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3841 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3901 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3961 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4021 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4081 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4141 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4201 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4261 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4321 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4381 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4441 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4501 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     4561 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4621 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4681 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4741 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4801 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4861 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4921 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4981 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5041 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5101 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5161 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5221 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5281 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5341 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5401 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5461 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5521 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5581 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5641 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5701 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5761 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5821 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     5881 gcgtatcacg aggccctttc gtc
//
