LOCUS       sequence-205212-        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   Ye Y, Lee IJ, Runge KW, Wu JQ
  TITLE     Roles of putative Rho-GEF Gef2 in division-site positioning and 
            contractile-ring function in fission yeast cytokinesis.
  JOURNAL   Mol Biol Cell. 2012 Apr;23(7):1181-95. doi: 10.1091/mbc.E11-09-0800.
            Epub 2012 Feb 1.
  PUBMED    22298427
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.E11-09-0800"; journalName: "Mol Biol Cell"; date: 
            "2012-04"; volume: "23"; issue: "7"; pages: "1181-95"
COMMENT     Sequence Label: pFA6a-kanMX6-P3nmt1-mECitrine
FEATURES             Location/Qualifiers
     source          1..6045
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(76..263)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     184..208
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(327..347)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     942..970
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     promoter        complement(1012..2175)
                     /label=nmt1 promoter
                     /note="wild-type nmt1 promoter from Schizosaccharomyces 
                     pombe, conferring strong thiamine-repressible expression"
     gene            2220..3576
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        2220..2563
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2564..3373
                     /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(2631..2650)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      3379..3576
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3680..3699)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3681..3699)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3786..3803)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3957..4545)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4037..4056)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4716..5507,5508..5576))
                     /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     5339..5358
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5577..5681)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5749..5767
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5805..5827)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5927..5946
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6027..6045
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     6027..6044
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 acgcggccgc cagctgaagc ttcgtacgct gcaggtcgac ggatctatat taccctgtta
       61 tccctagcgg atctgccggt agaggtgtgg tcaataagag cgacctcata ctatacctga
      121 gaaagcaacc tgacctacag gaaagagtta ctcaagaata agaattttcg ttttaaaacc
      181 taagagtcac tttaaaattt gtatacactt atttttttta taacttattt aataataaaa
      241 atcataaatc ataagaaatt cgcttattta gaagtggcgc gccctatttg tatagttcat
      301 ccatgccatg tgtaatccca gcagctgtta caaactcaag aaggaccatg tggtctctct
      361 tttcgttggg atctttcgaa agcttagatt gataggacag gtaatggttg tctggtaaaa
      421 ggacagggcc atcgccaatt ggagtatttt gttgataatg gtctgctagt tgaacgcttc
      481 catcttcaat gttgtgtcta attttgaagt taactttgat tccattcttt tgtttgtctg
      541 ccatgatgta tacattgtgt gagttatagt tgtattccaa tttgtgtcca agaatgtttc
      601 catcttcttt aaaatcaata ccttttaact cgattctatt aacaagggta tcaccttcaa
      661 acttgacttc agcacgtgtc ttgtagttcc cgtcatcttt gaaaaatata gttctttcct
      721 gtacataacc ttcgggcatg gcactcttga aaaagtcatg ccgtttcata tgatctgggt
      781 atctagcaaa gcacattaga ccataaccca aagtagtgac aagtgttggc catggaacag
      841 gtagttttcc agtagtgcaa ataaatttaa gggtaagttt tccgtatgtt gcatcacctt
      901 caccctctcc actgacagaa aatttgtgcc cattaacatc accatctaat tcaacaagaa
      961 ttgggacaac tccagtgaaa agttcttctc ctttactgtt aattaaagac atgatttaac
     1021 aaagcgacta taagtcagaa agtgagaatg agattgaaat aattaattca acttattcaa
     1081 ttgatgatat gccaggattc ctcttccttt atatatcttc aatgtttcga ttaacaggtc
     1141 cattatccgg tttttagttt cgctgcacat tgccgaatga caatcggatt cctttaagct
     1201 ttgcttttgc cctgttttct ctccccagta atcgttgggt gactgaacca tccagtagcc
     1261 gagagttacg tttttcctga tttaataaat atccatatct atttgtctcg tggtgtttta
     1321 cagcaactaa ccgaaattac tattttgcat catccaacca taaagagaca acaataattc
     1381 tagaaaacat ataacaagtg tttttttcta tttacgtctc atacgcaaag tccactttac
     1441 catgttttgg aaacttgtac ccaaccattc atttttcgat tttgtaatga ggctcttttc
     1501 ttctaacaat cattgaggtc attatcatgt gtcctcacct attgtttaaa ttgaggcttg
     1561 cgctattccc aaaattgtgg gtattatttt catatatcag tatctgcaat ttcggttcaa
     1621 ctttcttatc aagaaagagg attaatttct taacaaaaac cttctttgtt ttataacgct
     1681 gatttttgta ttatttggcg accaaggtta taagtgatcc aatccaaatg tcataacttc
     1741 agttgatccc tgcggaaaat ataaatcaga ttagacatat ggttatataa aaaaaaagag
     1801 aaattatttg agttcattga aacttcaacg ttatttgact aaaacttgtt aatttacatg
     1861 tctgcaagat taatccagca tattaaataa attagttgac ttatgtcaaa caaataatac
     1921 attcttgata ttattagaaa aaaaaagaaa attgaataac ttgtgagaaa tgatattgtt
     1981 attattttag attatgtcct ttgaaataca ataacatgct aaggcactta ctacttaagc
     2041 ctttattact ttaatgtgct aacaatcttc tgtatcctta gacgttaaat tttcccttat
     2101 taaaaaataa aaaaataaaa aaaaaatata ggaaatgaaa caaccgctga tgacttattc
     2161 tgtcttttat ggcgagatct gtttagcttg cctcgtcccc gccgggtcac ccggccagcg
     2221 acatggaggc ccagaatacc ctccttgaca gtcttgacgt gcgcagctca ggggcatgat
     2281 gtgactgtcg cccgtacatt tagcccatac atccccatgt ataatcattt gcatccatac
     2341 attttgatgg ccgcacggcg cgaagcaaaa attacggctc ctcgctgcag acctgcgagc
     2401 agggaaacgc tcccctcaca gacgcgttga attgtcccca cgccgcgccc ctgtagagaa
     2461 atataaaagg ttaggatttg ccactgaggt tcttctttca tatacttcct tttaaaatct
     2521 tgctaggata cagttctcac atcacatccg aacataaaca accatgggta aggaaaagac
     2581 tcacgtttcg aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg
     2641 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga
     2701 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga
     2761 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat
     2821 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccggcaaaa cagcattcca
     2881 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct
     2941 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg
     3001 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga
     3061 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataagc ttttgccatt
     3121 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga
     3181 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga
     3241 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt
     3301 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga
     3361 tgagtttttc taatcagtac tgacaataaa aagattcttg ttttcaagaa cttgtcattt
     3421 gtatagtttt tttatattgt agttgttcta ttttaatcaa atgttagcgt gatttatatt
     3481 ttttttcgcc tcgacatcat ctgcccagat gcgaagttaa gtgcgcagaa agtaatatca
     3541 tgcgtcaatc gtatgtgaat gctggtcgct atactgctgt cgattcgata ctaacgccgc
     3601 catccagttt aaacgagctc gaattcatcg atgatatcag atccactagt ggcctatgcg
     3661 gccgcggatc tgccggtctc cctatagtga gtcgtattaa tttcgataag ccaggttaac
     3721 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     3781 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3841 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3901 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3961 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4021 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4081 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4141 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4201 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4261 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4321 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4381 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4441 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4501 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4561 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4621 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4681 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4741 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4801 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     4861 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4921 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     4981 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5041 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5101 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5161 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5221 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5281 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5341 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5401 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5461 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5521 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5581 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5641 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5701 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     5761 acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     5821 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     5881 ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag
     5941 attgtactga gagtgcacca tatggacata ttgtcgttag aacgcggcta caattaatac
     6001 ataaccttat gtatcataca catacgattt aggtgacact ataga
//
