LOCUS       sequence-203604-        6045 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 6045)
  AUTHORS   Laporte D, Ojkic N, Vavylonis D, Wu JQ
  TITLE     alpha-Actinin and fimbrin cooperate with myosin II to organize 
            actomyosin bundles during contractile-ring assembly.
  JOURNAL   Mol Biol Cell. 2012 Aug;23(16):3094-110. doi: 
            10.1091/mbc.E12-02-0123. Epub 2012 Jun 27.
  PUBMED    22740629
REFERENCE   2  (bases 1 to 6045)
  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: 
            "10.1091/mbc.E12-02-0123"; journalName: "Mol Biol Cell"; date: 
            "2012-08"; volume: "23"; issue: "16"; pages: "3094-110"
COMMENT     Sequence Label: pFA6a-kanMX6-P3nmt1-mEGFP
FEATURES             Location/Qualifiers
     source          1..6045
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     15..33
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(71..93)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     193..212
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        293..311
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     293..310
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     terminator      complement(387..574)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     495..519
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(598..1308)
                     /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             complement(598..1308)
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLSPDNHYLSTQSKLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(638..658)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     1253..1281
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     promoter        complement(1323..2486)
                     /label=nmt1 promoter
                     /note="wild-type nmt1 promoter from Schizosaccharomyces 
                     pombe, conferring strong thiamine-repressible expression"
     gene            2531..3887
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        2531..2874
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2875..3684
                     /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(2942..2961)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      3690..3887
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3991..4010)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3992..4010)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4097..4114)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4268..4856)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4348..4367)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5027..5818,5819..5887))
                     /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     5650..5669
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5888..5992)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
       61 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
      121 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
      181 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg gacatattgt
      241 cgttagaacg cggctacaat taatacataa ccttatgtat catacacata cgatttaggt
      301 gacactatag aacgcggccg ccagctgaag cttcgtacgc tgcaggtcga cggatctata
      361 ttaccctgtt atccctagcg gatctgccgg tagaggtgtg gtcaataaga gcgacctcat
      421 actatacctg agaaagcaac ctgacctaca ggaaagagtt actcaagaat aagaattttc
      481 gttttaaaac ctaagagtca ctttaaaatt tgtatacact tatttttttt ataacttatt
      541 taataataaa aatcataaat cataagaaat tcgcttattt agaagtggcg cgccctattt
      601 gtatagttca tccatgccat gtgtaatccc agcagctgtt acaaactcaa gaaggaccat
      661 gtggtctctc ttttcgttgg gatctttcga aagcttagat tgtgtggaca ggtaatggtt
      721 gtctggtgaa aggacagggc catcgccaat tggagtattt tgttgataat ggtctgctag
      781 ttgaacgctt ccatcttcaa tgttgtgtct aattttgaag ttaactttga ttccattctt
      841 ttgtttgtct gccatgatgt atacattgtg tgagttatag ttgtattcca atttgtgtcc
      901 aagaatgttt ccatcttctt taaaatcaat accttttaac tcgattctat taacaagggt
      961 atcaccttca aacttgactt cagcacgtgt cttgtagttc ccgtcatctt tgaaaaatat
     1021 agttctttcc tgtacataac cttcgggcat ggcactcttg aaaaagtcat gctgtttcat
     1081 atgatctggg tatcttgaaa agcattgaac accataagtc agagtagtga caagtgttgg
     1141 ccatggaaca ggtagttttc cagtagtgca aataaattta agggtaagtt ttccgtatgt
     1201 tgcatcacct tcaccctctc cactgacaga aaatttgtgc ccattaacgt caccatctaa
     1261 ttcaacaaga attgggacaa ctccagtgaa aagttcttct cctttactgt taattaaaga
     1321 catgatttaa caaagcgact ataagtcaga aagtgagaat gagattgaaa taattaattc
     1381 aacttattca attgatgata tgccaggatt cctcttcctt tatatatctt caatgtttcg
     1441 attaacaggt ccattatccg gtttttagtt tcgctgcaca ttgccgaatg acaatcggat
     1501 tcctttaagc tttgcttttg ccctgttttc tctccccagt aatcgttggg tgactgaacc
     1561 atccagtagc cgagagttac gtttttcctg atttaataaa tatccatatc tatttgtctc
     1621 gtggtgtttt acagcaacta accgaaatta ctattttgca tcatccaacc ataaagagac
     1681 aacaataatt ctagaaaaca tataacaagt gtttttttct atttacgtct catacgcaaa
     1741 gtccacttta ccatgttttg gaaacttgta cccaaccatt catttttcga ttttgtaatg
     1801 aggctctttt cttctaacaa tcattgaggt cattatcatg tgtcctcacc tattgtttaa
     1861 attgaggctt gcgctattcc caaaattgtg ggtattattt tcatatatca gtatctgcaa
     1921 tttcggttca actttcttat caagaaagag gattaatttc ttaacaaaaa ccttctttgt
     1981 tttataacgc tgatttttgt attatttggc gaccaaggtt ataagtgatc caatccaaat
     2041 gtcataactt cagttgatcc ctgcggaaaa tataaatcag attagacata tggttatata
     2101 aaaaaaaaga gaaattattt gagttcattg aaacttcaac gttatttgac taaaacttgt
     2161 taatttacat gtctgcaaga ttaatccagc atattaaata aattagttga cttatgtcaa
     2221 acaaataata cattcttgat attattagaa aaaaaaagaa aattgaataa cttgtgagaa
     2281 atgatattgt tattatttta gattatgtcc tttgaaatac aataacatgc taaggcactt
     2341 actacttaag cctttattac tttaatgtgc taacaatctt ctgtatcctt agacgttaaa
     2401 ttttccctta ttaaaaaata aaaaaataaa aaaaaaatat aggaaatgaa acaaccgctg
     2461 atgacttatt ctgtctttta tggcgagatc tgtttagctt gcctcgtccc cgccgggtca
     2521 cccggccagc gacatggagg cccagaatac cctccttgac agtcttgacg tgcgcagctc
     2581 aggggcatga tgtgactgtc gcccgtacat ttagcccata catccccatg tataatcatt
     2641 tgcatccata cattttgatg gccgcacggc gcgaagcaaa aattacggct cctcgctgca
     2701 gacctgcgag cagggaaacg ctcccctcac agacgcgttg aattgtcccc acgccgcgcc
     2761 cctgtagaga aatataaaag gttaggattt gccactgagg ttcttctttc atatacttcc
     2821 ttttaaaatc ttgctaggat acagttctca catcacatcc gaacataaac aaccatgggt
     2881 aaggaaaaga ctcacgtttc gaggccgcga ttaaattcca acatggatgc tgatttatat
     2941 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat
     3001 gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
     3061 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
     3121 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggcaaa
     3181 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
     3241 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat
     3301 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
     3361 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataag
     3421 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
     3481 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac
     3541 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     3601 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     3661 ttgatgctcg atgagttttt ctaatcagta ctgacaataa aaagattctt gttttcaaga
     3721 acttgtcatt tgtatagttt ttttatattg tagttgttct attttaatca aatgttagcg
     3781 tgatttatat tttttttcgc ctcgacatca tctgcccaga tgcgaagtta agtgcgcaga
     3841 aagtaatatc atgcgtcaat cgtatgtgaa tgctggtcgc tatactgctg tcgattcgat
     3901 actaacgccg ccatccagtt taaacgagct cgaattcatc gatgatatca gatccactag
     3961 tggcctatgc ggccgcggat ctgccggtct ccctatagtg agtcgtatta atttcgataa
     4021 gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4081 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4141 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4201 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4261 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4321 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4381 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4441 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4501 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4561 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4621 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4681 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     4741 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4801 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4861 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4921 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4981 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     5041 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     5101 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     5161 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     5221 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     5281 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     5341 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     5401 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     5461 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     5521 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     5581 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     5641 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     5701 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5761 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5821 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5881 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5941 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     6001 cccgaaaagt gccacctgac gtctaagaaa ccattattat catga
//
