LOCUS       sequence-203664-        6033 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 6033)
  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 6033)
  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-P81nmt1-mCFP
FEATURES             Location/Qualifiers
     source          1..6033
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      34..622
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     523..542
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     776..793
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     880..899
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        880..898
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     gene            complement(1003..2359)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(1003..1200)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1206..2015)
                     /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     1929..1948
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2016..2359)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        2404..3555
                     /label=nmt1 P81 promoter
                     /note="mutant nmt1 promoter from Schizosaccharomyces pombe,
                     conferring weak thiamine-repressible expression"
     CDS             3570..4280
                     /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(3597..3625)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4220..4240
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      4304..4491
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4359..4383)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(4567..4585)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4568..4585)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4666..4685)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4785..4807
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4845..4863)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4931..5035
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5036..5104,5105..5896)
                     /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(5254..5273)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
       61 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
      121 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
      181 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
      241 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
      301 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
      361 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
      421 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
      481 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
      541 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
      601 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
      661 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
      721 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
      781 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
      841 gccgattcat taatgcaggt taacctggct tatcgaaatt aatacgactc actataggga
      901 gaccggcaga tccgcggccg cataggccac tagtggatct gatatcatcg atgaattcga
      961 gctcgtttaa actggatggc ggcgttagta tcgaatcgac agcagtatag cgaccagcat
     1021 tcacatacga ttgacgcatg atattacttt ctgcgcactt aacttcgcat ctgggcagat
     1081 gatgtcgagg cgaaaaaaaa tataaatcac gctaacattt gattaaaata gaacaactac
     1141 aatataaaaa aactatacaa atgacaagtt cttgaaaaca agaatctttt tattgtcagt
     1201 actgattaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     1261 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     1321 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     1381 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     1441 acgactgaat ccggtgagaa tggcaaaagc ttatgcattt ctttccagac ttgttcaaca
     1501 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     1561 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     1621 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     1681 ggatattctt ctaatacctg gaatgctgtt ttgccgggga tcgcagtggt gagtaaccat
     1741 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     1801 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     1861 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     1921 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     1981 cgcggcctcg aaacgtgagt cttttcctta cccatggttg tttatgttcg gatgtgatgt
     2041 gagaactgta tcctagcaag attttaaaag gaagtatatg aaagaagaac ctcagtggca
     2101 aatcctaacc ttttatattt ctctacaggg gcgcggcgtg gggacaattc aacgcgtctg
     2161 tgaggggagc gtttccctgc tcgcaggtct gcagcgagga gccgtaattt ttgcttcgcg
     2221 ccgtgcggcc atcaaaatgt atggatgcaa atgattatac atggggatgt atgggctaaa
     2281 tgtacgggcg acagtcacat catgcccctg agctgcgcac gtcaagactg tcaaggaggg
     2341 tattctgggc ctccatgtcg ctggccgggt gacccggcgg ggacgaggca agctaaacag
     2401 atctcgccat aaaagacaga ataagtcatc agcggttgtt tcatttccta tatttttttt
     2461 ttattttttt attttttaat aagggaaaat ttaacgtcta aggatacaga agattgttag
     2521 cacattaaag taataaaggc ttaagtagta agtgccttag catgttattg tatttcaaag
     2581 gacataatct aaaataataa caatatcatt tctcacaagt tattcaattt tctttttttt
     2641 ttctaataat atcaagaatg tattatttgt ttgacataag tcaactaatt tatttaatat
     2701 gctggattaa tcttgcagac atgtaaatta acaagtttta gtcaaataac gttgaagttt
     2761 caatgaactc aaataatttc tctttttttt tatataacca taatctgatt tatattttcc
     2821 gcagggatca actgaagtta tgacatttgg attggatcac ttataacctt ggtcgccaaa
     2881 taatacaaaa atcagcgtta taaaacaaag aaggtttttg ttaagaaatt aatcctcttt
     2941 cttgataaga aagttgaacc gaaattgcag atactgatat atgaaaataa tacccacaat
     3001 tttgggaata gcgcaagcct caatttaaac aataggtgag gacacatgat aatgacctca
     3061 atgattgtta gaagaaaaga gcctcattac aaaatcgaaa aatgaatggt tgggtacaag
     3121 tttccaaaac atggtaaagt ggactttgcg tatgagacgt aaatagaaaa aaacacttgt
     3181 tatatgtttt ctagaattat tgttgtctct ttatggttgg atgatgcaaa atagtaattt
     3241 cggttagttg ctgtaaaaca ccacgagaca aatagatatg gatatttatt aaatcaggaa
     3301 aaacgtaact ctcggctact ggatggttca gtcacccaac gattactggg gagagaaaac
     3361 agggcaaaag caaagcttaa aggaatccga ttgtcattcg gcaatgtgca gcgaaactaa
     3421 aaaccggata atggacctgt taatcgaaac attgaagatg gaagaggaat cctggcatat
     3481 catcaattga ataagttgaa ttaattattt caatctcatt ctcactttct gacttatagt
     3541 cgctttgtta aatcatgtct ttaattaaca gtaaaggaga agaacttttc actggagttg
     3601 tcccaattct tgttgaatta gatggtgatg ttaatgggca caaattttct gtcagtggag
     3661 agggtgaagg tgatgcaaca tacggaaaac ttacccttaa atttatttgc actactggaa
     3721 aactacctgt tccatggcca acacttgtca ctactttgac ttggggtgtt caatgctttt
     3781 caagataccc agatcatatg aaacggcatg actttttcaa gagtgccatg cccgaaggtt
     3841 atgtacagga aagaactata tttttcaaag atgacgggaa ctacaagaca cgtgctgaag
     3901 tcaagtttga aggtgatacc cttgttaata gaatcgagtt aaaaggtatt gattttaaag
     3961 aagatggaaa cattcttgga cacaaattgg aatacaacta tatttcacac aatgtataca
     4021 tcactgcaga caaacaaaag aatggaatca aagctcattt caaaattaga cacaacattg
     4081 aagatggaag cgttcaacta gcagaccatt atcaacaaaa tactccaatt ggcgatggcc
     4141 ctgtcctttt accagacaac cattacctgt ccacacaatc taagctttcg aaagatccca
     4201 acgaaaagag agaccacatg gtccttcttg agtttgtaac agctgctggg attacacatg
     4261 gcatggatga actatacaaa tagggcgcgc cacttctaaa taagcgaatt tcttatgatt
     4321 tatgattttt attattaaat aagttataaa aaaaataagt gtatacaaat tttaaagtga
     4381 ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac tctttcctgt aggtcaggtt
     4441 gctttctcag gtatagtatg aggtcgctct tattgaccac acctctaccg gcagatccgc
     4501 tagggataac agggtaatat agatccgtcg acctgcagcg tacgaagctt cagctggcgg
     4561 ccgcgttcta tagtgtcacc taaatcgtat gtgtatgata cataaggtta tgtattaatt
     4621 gtagccgcgt tctaacgaca atatgtccat atggtgcact ctcagtacaa tctgctctga
     4681 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     4741 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     4801 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     4861 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     4921 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     4981 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     5041 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5101 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5161 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5221 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     5281 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5341 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5401 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     5461 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     5521 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     5581 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     5641 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     5701 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     5761 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     5821 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     5881 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     5941 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6001 aatcccttaa cgtgagtttt cgttccactg agc
//
