LOCUS       sequence-39186-e        5216 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  genomic GFP tagging in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5216)
  AUTHORS   Gadaleta MC, Iwasaki O, Noguchi C, Noma K, Noguchi E
  TITLE     New vectors for epitope tagging and gene disruption in 
            Schizosaccharomyces pombe.
  JOURNAL   Biotechniques. 2013 Nov;55(5):257-63. doi: 10.2144/000114100.
  PUBMED    24215641
REFERENCE   2  (bases 1 to 5216)
  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"; doi: "10"; journalName: 
            "Biotechniques"; date: "2013-11"; volume: "55"; issue: "5"; pages: 
            "257-63"
COMMENT     Sequence Label: pFA6a-GFP-ura4MX6
FEATURES             Location/Qualifiers
     source          1..5216
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             63..773
                     /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="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(90..118)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     713..733
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      797..984
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(852..876)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(1453..2247)
                     /codon_start=1
                     /gene="Schizosaccharomyces pombe ura4"
                     /product="orotidine 5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=S. pombe ura4
                     /translation="MDARVFQSYSARAEGMKNPIAKELLALMEEKQSNLSVAVDLTKKS
                     EILELVDKIGPYVCVIKTHIDVVEDFDQDMVEKLVALGKKHRFLIFEDRKFADIGNTVK
                     LQYASGVYKIASWAHITNCHTVPGEGIIQGLKEVGLPLGRGLLLLAEMSSKGSLATGSY
                     TEKTLEWFEKHTDFCFGFIAGRRFPNLQSDYITMSPGIGLDVKGDGLGQQYRTPEEVIV
                     NCGSDIIIVGRGVYGAGRNPVVEAKRYREAGWKAYQQRLSQH"
     primer_bind     complement(2853..2872)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2854..2872)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2959..2976)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3130..3718)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3210..3229)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3889..4680,4681..4749))
                     /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     4512..4531
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4750..4854)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4922..4940
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4978..5000)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5100..5119
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(5200..5216,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(5200..5216,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg acggatcccc gggttaatta
       61 acagtaaagg agaagaactt ttcactggag ttgtcccaat tcttgttgaa ttagatggtg
      121 atgttaatgg gcacaaattt tctgtcagtg gagagggtga aggtgatgca acatacggaa
      181 aacttaccct taaatttatt tgcactactg gaaaactacc tgttccatgg ccaacacttg
      241 tcactacttt cacttatggt gttcaatgct tttcaagata cccagatcat atgaaacggc
      301 atgacttttt caagagtgcc atgcccgaag gttatgtaca ggaaagaact atatttttca
      361 aagatgacgg gaactacaag acacgtgctg aagtcaagtt tgaaggtgat acccttgtta
      421 atagaatcga gttaaaaggt attgatttta aagaagatgg aaacattctt ggacacaaat
      481 tggaatacaa ctataactca cacaatgtat acatcatggc agacaaacaa aagaatggaa
      541 tcaaagttaa cttcaaaatt agacacaaca ttgaagatgg aagcgttcaa ctagcagacc
      601 attatcaaca aaatactcca attggcgatg gccctgtcct tttaccagac aaccattacc
      661 tgtccacaca atctgccctt tcgaaagatc ccaacgaaaa gagagaccac atggtccttc
      721 ttgagtttgt aacagctgct gggattacac atggcatgga tgaactatac aaatagggcg
      781 cgccacttct aaataagcga atttcttatg atttatgatt tttattatta aataagttat
      841 aaaaaaaata agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt
      901 attcttgagt aactctttcc tgtaggtcag gttgctttct caggtatagt atgaggtcgc
      961 tcttattgac cacacctcta ccggcagatc cgctagggat aacagggtaa tatagatctc
     1021 ttgtgatatt gacgaaactt tttgacatct aatttattct gttccaacac caatgtttat
     1081 aaccaagttt tatcttgttt gtctacatgg tattttacat tcatctacat acatctttca
     1141 ttggctttgt acatagttat cattacaagt ctaaaaaaat tcactctttt cttattcaat
     1201 gtcaatccaa gagaaaagat tgtggtaatg ttgtaggagc atgtttaata aattactata
     1261 gcaaattact ttttattccc aaggtgttta tctataatag ttaatatttt agtcgctaca
     1321 taaaatttta ccaaagagta cttgtatact aattctaaat gccttctgac ataaaacgcc
     1381 taggaaaaca aacgcaaaca aggcatcgac tttttcaata accaaccaaa aaaattttac
     1441 attagtcttt ttttaatgct gagaaagtct ttgctgatat gccttccaac cagcttctct
     1501 atatctcttg gcttcgacaa caggattacg accagctcca tagactccac gaccaacaat
     1561 gatgatatcg ctaccgcagt ttacaatcac ttcttcagga gtacgatatt gctgtcccag
     1621 cccgtctcct ttaacatcca agccgatacc aggggacata gttatgtagt cgctttgaag
     1681 gttaggaaat cgacgaccag ctataaagcc aaagcaaaaa tcggtatgct tctcaaacca
     1741 ttctaaggtt ttctctgtgt aggaaccagt agccaaagag cctttggaag acatttcagc
     1801 caaaagcaag agaccacgtc ccaaaggtaa accaacttct ttgaggcctt gtataatacc
     1861 ctcgcctggc actgtatggc aatttgtgat atgagcccaa gaagcaattt tgtacacacc
     1921 agatgcatat tgtagcttga cggtatttcc aatgtctgcg aatttgcgat cctcaaagat
     1981 aagaaaacga tgctttttac ctaaggccac cagtttttct accatatcct ggtcgaaatc
     2041 ctcgacaacg tcaatatgtg tcttgataac acagacatag ggtccaattt tatctaccaa
     2101 ttctaagatt tcggatttct tcgtcaaatc gaccgcgact gacaagttgc tttgcttttc
     2161 ttccatcaaa gccaacaatt ccttggcaat gggatttttc atcccctcag ctctagctga
     2221 atagctttga aatactctag catccataac tttgctttta aacctttaat ttcgatccaa
     2281 gcaaaaaaga ggttcttggt aggacaatac ggtaagaaaa cacgacatgt gcagagatgc
     2341 cgacgaagca tagttaaact gggatggtaa aatcaattaa gaatttataa agacaaaatt
     2401 gtataagtct ctaaaacatc ttaattatac ctcacagaac tatctaaaat atattcacaa
     2461 agtgcaaaca ttatcatgaa aaagaaccat tttaatttaa agcaagggca ttaaggctta
     2521 tttacagaat ttcttacttt tgtaaagatt ataaggctga ttatcttttt caccatgcca
     2581 aaaattacac aagatagaat ggatgtttga aattaaacgt gagtatacaa acaaatacac
     2641 taggtaaatc gaaacatttt tttctccatt aagtaacaaa ttcctattta gagaaagaat
     2701 gctgagtaga ttaaataatc tatacaaact tttttaacac aaatgcatac atatagccag
     2761 tgggatttgt agctaagctg tttaaacgag ctcgaattca tcgatgatat cagatccact
     2821 agtggcctat gcggccgcgg atctgccggt ctccctatag tgagtcgtat taatttcgat
     2881 aagccaggtt aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     2941 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     3001 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     3061 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     3121 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     3181 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     3241 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     3301 ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt
     3361 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     3421 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     3481 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     3541 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     3601 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3661 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     3721 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     3781 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     3841 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     3901 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     3961 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     4021 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     4081 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     4141 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     4201 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     4261 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     4321 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     4381 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     4441 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     4501 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     4561 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     4621 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     4681 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     4741 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     4801 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     4861 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
     4921 aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct
     4981 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     5041 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc
     5101 ggcatcagag cagattgtac tgagagtgca ccatatggac atattgtcgt tagaacgcgg
     5161 ctacaattaa tacataacct tatgtatcat acacatacga tttaggtgac actata
//
