LOCUS       sequence-83386-e        6369 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 6369)
  AUTHORS   Ito-Harashima S, McCusker JH
  TITLE     Positive and negative selection LYS5MX gene replacement cassettes 
            for use in Saccharomyces cerevisiae.
  JOURNAL   Yeast. 2004 Jan 15;21(1):53-61.
  PUBMED    14745782
REFERENCE   2  (bases 1 to 6369)
  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: "Yeast."; 
            date: "2004-01-15"; volume: "21(1)"; pages: "53-61"
COMMENT     Sequence Label: pSA25
FEATURES             Location/Qualifiers
     source          1..6369
                     /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        190..410
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             411..1214
                     /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(1343..1798)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1480..1501)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1692..1711
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1928..1950
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1942..1959
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1943..1959
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1966..1985
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1966..1984
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(2101..2298)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     promoter        complement(3123..3466)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        complement(3595..3613)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3634..3650)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3634..3650)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3647..3669)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3658..3674
                     /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(3682..3688,3689..3706,3707..3712))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3865..3882)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4036..4624)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4795..5586,5587..5655))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5418..5437
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5656..5760)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    5797..6300
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     6342..6360
                     /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 accacgcttt tcaattcaat tcatcatttt ttttttattc ttttttttga tttcggtttc
      241 tttgaaattt ttttgattcg gtaatctccg aacagaagga agaacgaagg aaggagcaca
      301 gacttagatt ggtatatata cgcatatgta gtgttgaaga aacatgaaat tgcccagtat
      361 tcttaaccca actgcacaga acaaaaacct gcaggaaacg aagataaatc atgtcgaaag
      421 ctacatataa ggaacgtgct gctactcatc ctagtcctgt tgctgccaag ctatttaata
      481 tcatgcacga aaagcaaaca aacttgtgtg cttcattgga tgttcgtacc accaaggaat
      541 tactggagtt agttgaagca ttaggtccca aaatttgttt actaaaaaca catgtggata
      601 tcttgactga tttttccatg gagggcacag ttaagccgct aaaggcatta tccgccaagt
      661 acaatttttt actcttcgaa gacagaaaat ttgctgacat tggtaataca gtcaaattgc
      721 agtactctgc gggtgtatac agaatagcag aatgggcaga cattacgaat gcacacggtg
      781 tggtgggccc aggtattgtt agcggtttga agcaggcggc agaagaagta acaaaggaac
      841 ctagaggcct tttgatgtta gcagaattgt catgcaaggg ctccctatct actggagaat
      901 atactaaggg tactgttgac attgcgaaga gcgacaaaga ttttgttatc ggctttattg
      961 ctcaaagaga catgggtgga agagatgaag gttacgattg gttgattatg acacccggtg
     1021 tgggtttaga tgacaaggga gacgcattgg gtcaacagta tagaaccgtg gatgatgtgg
     1081 tctctacagg atctgacatt attattgttg gaagaggact atttgcaaag ggaagggatg
     1141 ctaaggtaga gggtgaacgt tacagaaaag caggctggga agcatatttg agaagatgcg
     1201 gccagcaaaa ctaaaaaact gtattataag taaatgcatg tatactaaac tcacaaatta
     1261 gagcttcaat ttaattatat cagttattac cctgcggtgt gaaataccgc acagatgcgt
     1321 aaggagaaaa taccgcatca ggaaattgta aacgttaata ttttgttaaa attcgcgtta
     1381 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     1441 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     1501 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     1561 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     1621 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     1681 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     1741 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcg
     1801 cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     1861 ctattacgcc agctggcgaa ggggggatgt gctgcaaggc gattaagttg ggtaacgcca
     1921 gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta
     1981 tagggcgaat tggagctcca ccgcggtggc ggccgcatag gccactagtg gatctgatat
     2041 catcgatgaa ttcgagctcg ttttcgacac tggatggcgg cgttagtatc gaatcgacag
     2101 cagtatagcg accagcattc acatacgatt gacgcatgat attactttct gcgcacttaa
     2161 cttcgcatct gggcagatga tgtcgaggcg aaaaaaaata taaatcacgc taacatttga
     2221 ttaaaataga acaactacaa tataaaaaaa ctatacaaat gacaagttct tgaaaacaag
     2281 aatcttttta ttgtcagtac tgattataaa ccatcatttt cgatgaaata atcaatcaac
     2341 gtagataagc tgatattata taattttggt ctgttcgtgt tgattttatc actgatggac
     2401 tttggcatac agatagtgac aatttcgtta ttgaaccatt gagaatggaa aatcaatgga
     2461 acttcatcca gagttatgca catagaagct ccctcagccg gaaaaaagct gatagcgcca
     2521 aaatctatta gtgacaagtc tgtgttaagg ccagttccag taaattttgt atacgactcc
     2581 ttcaaggacc ataagtaagt aaatattgtg catggatcag acgctttcag taaaccgtta
     2641 aattctcttt cactaaaaac ttctttaaat agctccaact cttccctccc gccataattg
     2701 cacggagaag cgatatcaat tccgacatcc tggtattcat ctgtacttac acattttacg
     2761 aggaacatag ctacatattg ttcaccgatg gtcatgctaa atggaagaaa acgattgttg
     2821 tctaagaatg gcttaccgaa gctgcccttg tcaaatttca gctcttgaaa atttaagccc
     2881 gttactatag agcagccaaa caactgcagc agctggctgc atagatttga acatctatcg
     2941 tgaaacgatt ttttattgag gattctggct tgagacgcca atggcaaagt tctcattaat
     3001 gcctcgaacg taaactcatc cgcgagtata tcctcttgaa tttcaacaac gaatatacct
     3061 gcccatggtc ttacacctgc cacctttgaa acttcgctta ctacttcagt cgttttaacc
     3121 atggttgttt atgttcggat gtgatgtgag aactgtatcc tagcaagatt ttaaaaggaa
     3181 gtatatgaaa gaagaacctc agtggcaaat cctaaccttt tatatttctc tacaggggcg
     3241 cggcgtgggg acaattcaac gcgtctgtga ggggagcgtt tccctgctcg caggtctgca
     3301 gcgaggagcc gtaatttttg cttcgcgccg tgcggccatc aaaatgtatg gatgcaaatg
     3361 attatacatg gggatgtatg ggctaaatgt acgggcgaca gtcacatcat gcccctgagc
     3421 tgcgcacgtc aagactgtca aggagggtat tctgggcctc catgtcgctg gccgggtgac
     3481 ccggcgggga caaggcaagc taaacagatc tggcgcgcct taattaaccc ggggatccgt
     3541 cgacctgcag cgtacgaagc ttcagctggc ggccgctcta gccagctttt gttcccttta
     3601 gtgagggtta attccgagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     3661 ttatccgctc acaattccac acaacatagg agccggaagc ataaagtgta aagcctgggg
     3721 tgcctaatga gtgaggtaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     3781 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3841 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     3901 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     3961 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4021 cgcgttgctg gcgtttttcc ataggctcgg cccccctgac gagcatcaca aaaatcgacg
     4081 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt tcccccctgg
     4141 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4201 tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt
     4261 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4321 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4381 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4441 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     4501 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4561 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4621 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4681 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     4741 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     4801 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     4861 ctgactgccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     4921 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     4981 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5041 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5101 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5161 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgaaaaa aagcggttag
     5221 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     5281 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     5341 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     5401 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5461 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     5521 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     5581 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     5641 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     5701 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     5761 cacatttccc cgaaaagtgc cacctgggtc cttttcatca cgtgctataa aaataattat
     5821 aatttaaatt ttttaatata aatatataaa ttaaaaatag aaagtaaaaa aagaaattaa
     5881 agaaaaaata gtttttgttt tccgaagatg taaaagactc tagggggatc gccaacaaat
     5941 actacctttt atcttgctct tcctgctctc aggtattaat gccgaattgt ttcatcttgt
     6001 ctgtgtagaa gaccacacac gaaaatcctg tgattttaca ttttacttat cgttaatcga
     6061 atgtatatct atttaatctg cttttcttgt ctaataaata tatatgtaaa gtacgctttt
     6121 tgttgaaatt ttttaaacct ttgtttattt ttttttcttc attccgtaac tcttctacct
     6181 tctttattta ctttctaaaa tccaaataca aaacataaaa ataaataaac acagagtaaa
     6241 ttcccaaatt attccatcat taaaagatac gaggcgcgtg taagttacag gcaagcgatc
     6301 cgtcctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     6361 cctttcgtc
//
