LOCUS       sequence-203648-        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   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 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"; journalName: "Dev Cell.";
            date: "2003-11"; volume: "5(5)"; pages: "723-34"
COMMENT     Sequence Label: pFA6a-kanMX6-P3nmt1-mCFP
FEATURES             Location/Qualifiers
     source          1..6045
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      24..612
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     513..532
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     766..783
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     870..889
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        870..888
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     gene            complement(993..2349)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(993..1190)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1196..2005)
                     /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     1919..1938
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2006..2349)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        2394..3557
                     /label=nmt1 promoter
                     /note="wild-type nmt1 promoter from Schizosaccharomyces 
                     pombe, conferring strong thiamine-repressible expression"
     CDS             3572..4282
                     /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
                     ICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKAH
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(3599..3627)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4222..4242
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      4306..4493
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4361..4385)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(4569..4587)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4570..4587)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4668..4687)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4787..4809
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4847..4865)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4933..5037
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5038..5106,5107..5898)
                     /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     complement(5256..5275)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
       61 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
      121 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
      181 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
      241 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
      301 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
      361 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
      421 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
      481 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
      541 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
      601 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
      661 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
      721 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
      781 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
      841 taatgcaggt taacctggct tatcgaaatt aatacgactc actataggga gaccggcaga
      901 tccgcggccg cataggccac tagtggatct gatatcatcg atgaattcga gctcgtttaa
      961 actggatggc ggcgttagta tcgaatcgac agcagtatag cgaccagcat tcacatacga
     1021 ttgacgcatg atattacttt ctgcgcactt aacttcgcat ctgggcagat gatgtcgagg
     1081 cgaaaaaaaa tataaatcac gctaacattt gattaaaata gaacaactac aatataaaaa
     1141 aactatacaa atgacaagtt cttgaaaaca agaatctttt tattgtcagt actgattaga
     1201 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     1261 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     1321 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     1381 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     1441 ccggtgagaa tggcaaaagc ttatgcattt ctttccagac ttgttcaaca ggccagccat
     1501 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     1561 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     1621 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     1681 ctaatacctg gaatgctgtt ttgccgggga tcgcagtggt gagtaaccat gcatcatcag
     1741 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     1801 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     1861 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     1921 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg
     1981 aaacgtgagt cttttcctta cccatggttg tttatgttcg gatgtgatgt gagaactgta
     2041 tcctagcaag attttaaaag gaagtatatg aaagaagaac ctcagtggca aatcctaacc
     2101 ttttatattt ctctacaggg gcgcggcgtg gggacaattc aacgcgtctg tgaggggagc
     2161 gtttccctgc tcgcaggtct gcagcgagga gccgtaattt ttgcttcgcg ccgtgcggcc
     2221 atcaaaatgt atggatgcaa atgattatac atggggatgt atgggctaaa tgtacgggcg
     2281 acagtcacat catgcccctg agctgcgcac gtcaagactg tcaaggaggg tattctgggc
     2341 ctccatgtcg ctggccgggt gacccggcgg ggacgaggca agctaaacag atctcgccat
     2401 aaaagacaga ataagtcatc agcggttgtt tcatttccta tatttttttt ttattttttt
     2461 attttttaat aagggaaaat ttaacgtcta aggatacaga agattgttag cacattaaag
     2521 taataaaggc ttaagtagta agtgccttag catgttattg tatttcaaag gacataatct
     2581 aaaataataa caatatcatt tctcacaagt tattcaattt tctttttttt tctaataata
     2641 tcaagaatgt attatttgtt tgacataagt caactaattt atttaatatg ctggattaat
     2701 cttgcagaca tgtaaattaa caagttttag tcaaataacg ttgaagtttc aatgaactca
     2761 aataatttct cttttttttt atataaccat atgtctaatc tgatttatat tttccgcagg
     2821 gatcaactga agttatgaca tttggattgg atcacttata accttggtcg ccaaataata
     2881 caaaaatcag cgttataaaa caaagaaggt ttttgttaag aaattaatcc tctttcttga
     2941 taagaaagtt gaaccgaaat tgcagatact gatatatgaa aataataccc acaattttgg
     3001 gaatagcgca agcctcaatt taaacaatag gtgaggacac atgataatga cctcaatgat
     3061 tgttagaaga aaagagcctc attacaaaat cgaaaaatga atggttgggt acaagtttcc
     3121 aaaacatggt aaagtggact ttgcgtatga gacgtaaata gaaaaaaaca cttgttatat
     3181 gttttctaga attattgttg tctctttatg gttggatgat gcaaaatagt aatttcggtt
     3241 agttgctgta aaacaccacg agacaaatag atatggatat ttattaaatc aggaaaaacg
     3301 taactctcgg ctactggatg gttcagtcac ccaacgatta ctggggagag aaaacagggc
     3361 aaaagcaaag cttaaaggaa tccgattgtc attcggcaat gtgcagcgaa actaaaaacc
     3421 ggataatgga cctgttaatc gaaacattga agatatataa aggaagagga atcctggcat
     3481 atcatcaatt gaataagttg aattaattat ttcaatctca ttctcacttt ctgacttata
     3541 gtcgctttgt taaatcatgt ctttaattaa cagtaaagga gaagaacttt tcactggagt
     3601 tgtcccaatt cttgttgaat tagatggtga tgttaatggg cacaaatttt ctgtcagtgg
     3661 agagggtgaa ggtgatgcaa catacggaaa acttaccctt aaatttattt gcactactgg
     3721 aaaactacct gttccatggc caacacttgt cactactttg acttggggtg ttcaatgctt
     3781 ttcaagatac ccagatcata tgaaacggca tgactttttc aagagtgcca tgcccgaagg
     3841 ttatgtacag gaaagaacta tatttttcaa agatgacggg aactacaaga cacgtgctga
     3901 agtcaagttt gaaggtgata cccttgttaa tagaatcgag ttaaaaggta ttgattttaa
     3961 agaagatgga aacattcttg gacacaaatt ggaatacaac tatatttcac acaatgtata
     4021 catcactgca gacaaacaaa agaatggaat caaagctcat ttcaaaatta gacacaacat
     4081 tgaagatgga agcgttcaac tagcagacca ttatcaacaa aatactccaa ttggcgatgg
     4141 ccctgtcctt ttaccagaca accattacct gtccacacaa tctaagcttt cgaaagatcc
     4201 caacgaaaag agagaccaca tggtccttct tgagtttgta acagctgctg ggattacaca
     4261 tggcatggat gaactataca aatagggcgc gccacttcta aataagcgaa tttcttatga
     4321 tttatgattt ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt
     4381 gactcttagg ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg
     4441 ttgctttctc aggtatagta tgaggtcgct cttattgacc acacctctac cggcagatcc
     4501 gctagggata acagggtaat atagatccgt cgacctgcag cgtacgaagc ttcagctggc
     4561 ggccgcgttc tatagtgtca cctaaatcgt atgtgtatga tacataaggt tatgtattaa
     4621 ttgtagccgc gttctaacga caatatgtcc atatggtgca ctctcagtac aatctgctct
     4681 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4741 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     4801 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     4861 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     4921 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     4981 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5041 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5101 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5161 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5221 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5281 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5341 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5401 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5461 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5521 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5581 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5641 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5701 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     5761 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     5821 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     5881 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     5941 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6001 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag acccc
//
