LOCUS       sequence-203672-        4882 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast gene targeting.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4882)
  AUTHORS   Wu JQ, Kuhn JR, Kovar DR, Pollard TD
  TITLE     Spatial and temporal pathway for assembly and constriction of the 
            contractile ring in fission yeast cytokinesis.
  JOURNAL   Dev Cell. 2003 Nov;5(5):723-34.
  PUBMED    14602073
REFERENCE   2  (bases 1 to 4882)
  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: "Dev Cell.";
            date: "2003-11"; volume: "5(5)"; pages: "723-34"
COMMENT     Sequence Label: pFA6a- mEGFP-kanMX6
FEATURES             Location/Qualifiers
     source          1..4882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     24..43
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        124..142
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     124..141
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             203..913
                     /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
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLSPDNHYLSTQSKLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             203..913
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLSPDNHYLSTQSKLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(230..258)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     853..873
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      937..1124
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(992..1016)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            1199..2555
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        1199..1542
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1543..2352
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1610..1629)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2358..2555
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2659..2678)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2660..2678)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2765..2782)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2936..3524)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3016..3035)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3695..4486,4487..4555))
                     /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     4318..4337
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4556..4660)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4728..4746
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4784..4806)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
       61 ggacatattg tcgttagaac gcggctacaa ttaatacata accttatgta tcatacacat
      121 acgatttagg tgacactata gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg
      181 acggatcccc gggttaatta acagtaaagg agaagaactt ttcactggag ttgtcccaat
      241 tcttgttgaa ttagatggtg acgttaatgg gcacaaattt tctgtcagtg gagagggtga
      301 aggtgatgca acatacggaa aacttaccct taaatttatt tgcactactg gaaaactacc
      361 tgttccatgg ccaacacttg tcactactct gacttatggt gttcaatgct tttcaagata
      421 cccagatcat atgaaacagc atgacttttt caagagtgcc atgcccgaag gttatgtaca
      481 ggaaagaact atatttttca aagatgacgg gaactacaag acacgtgctg aagtcaagtt
      541 tgaaggtgat acccttgtta atagaatcga gttaaaaggt attgatttta aagaagatgg
      601 aaacattctt ggacacaaat tggaatacaa ctataactca cacaatgtat acatcatggc
      661 agacaaacaa aagaatggaa tcaaagttaa cttcaaaatt agacacaaca ttgaagatgg
      721 aagcgttcaa ctagcagacc attatcaaca aaatactcca attggcgatg gccctgtcct
      781 ttcaccagac aaccattacc tgtccacaca atctaagctt tcgaaagatc ccaacgaaaa
      841 gagagaccac atggtccttc ttgagtttgt aacagctgct gggattacac atggcatgga
      901 tgaactatac aaatagggcg cgccacttct aaataagcga atttcttatg atttatgatt
      961 tttattatta aataagttat aaaaaaaata agtgtataca aattttaaag tgactcttag
     1021 gttttaaaac gaaaattctt attcttgagt aactctttcc tgtaggtcag gttgctttct
     1081 caggtatagt atgaggtcgc tcttattgac cacacctcta ccggcagatc cgctagggat
     1141 aacagggtaa tatagatctg tttagcttgc ctcgtccccg ccgggtcacc cggccagcga
     1201 catggaggcc cagaataccc tccttgacag tcttgacgtg cgcagctcag gggcatgatg
     1261 tgactgtcgc ccgtacattt agcccataca tccccatgta taatcatttg catccataca
     1321 ttttgatggc cgcacggcgc gaagcaaaaa ttacggctcc tcgctgcaga cctgcgagca
     1381 gggaaacgct cccctcacag acgcgttgaa ttgtccccac gccgcgcccc tgtagagaaa
     1441 tataaaaggt taggatttgc cactgaggtt cttctttcat atacttcctt ttaaaatctt
     1501 gctaggatac agttctcaca tcacatccga acataaacaa ccatgggtaa ggaaaagact
     1561 cacgtttcga ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg
     1621 gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat
     1681 gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag
     1741 atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc
     1801 cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggcaaaac agcattccag
     1861 gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg
     1921 cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt
     1981 ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac
     2041 gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataagct tttgccattc
     2101 tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag
     2161 gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat
     2221 cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt
     2281 caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat
     2341 gagtttttct aatcagtact gacaataaaa agattcttgt tttcaagaac ttgtcatttg
     2401 tatagttttt ttatattgta gttgttctat tttaatcaaa tgttagcgtg atttatattt
     2461 tttttcgcct cgacatcatc tgcccagatg cgaagttaag tgcgcagaaa gtaatatcat
     2521 gcgtcaatcg tatgtgaatg ctggtcgcta tactgctgtc gattcgatac taacgccgcc
     2581 atccagttta aacgagctcg aattcatcga tgatatcaga tccactagtg gcctatgcgg
     2641 ccgcggatct gccggtctcc ctatagtgag tcgtattaat ttcgataagc caggttaacc
     2701 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     2761 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     2821 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     2881 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     2941 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3001 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3061 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3121 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3181 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3241 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3301 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3361 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3421 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3481 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3541 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3601 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3661 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3721 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     3781 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     3841 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     3901 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     3961 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4021 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4081 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4141 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4201 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4261 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4321 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4381 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4441 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4501 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4561 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4621 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4681 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     4741 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     4801 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     4861 gcgcgtcagc gggtgttggc gg
//
