LOCUS       sequence-203268-        5310 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 5310)
  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 5310)
  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-hphMX6-41nmt1
FEATURES             Location/Qualifiers
     source          1..5310
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        14..32
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     14..31
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        complement(97..1251)
                     /label=nmt1 P41 promoter
                     /note="mutant nmt1 promoter from Schizosaccharomyces pombe,
                     conferring medium strength thiamine-repressible expression"
     gene            1296..2871
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        1296..1639
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1640..2668
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLMHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      2674..2871
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2977..2996)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2978..2996)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3083..3100)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3254..3842)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3334..3353)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4013..4804,4805..4873))
                     /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     4636..4655
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4874..4978)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5046..5064
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5102..5124)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5224..5243
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tcatacacat acgatttagg tgacactata gaacgcggcc gccagctgaa gcttcgtacg
       61 ctgcaggtcg acggatcccc gggttaatta aagacatgat ttaacaaagc gactataagt
      121 cagaaagtga gaatgagatt gaaataatta attcaactta ttcaattgat gatatgccag
      181 gattcctctt cctttatctt caatgtttcg attaacaggt ccattatccg gtttttagtt
      241 tcgctgcaca ttgccgaatg acaatcggat tcctttaagc tttgcttttg ccctgttttc
      301 tctccccagt aatcgttggg tgactgaacc atccagtagc cgagagttac gtttttcctg
      361 atttaataaa tatccatatc tatttgtctc gtggtgtttt acagcaacta accgaaatta
      421 ctattttgca tcatccaacc ataaagagac aacaataatt ctagaaaaca tataacaagt
      481 gtttttttct atttacgtct catacgcaaa gtccacttta ccatgttttg gaaacttgta
      541 cccaaccatt catttttcga ttttgtaatg aggctctttt cttctaacaa tcattgaggt
      601 cattatcatg tgtcctcacc tattgtttaa attgaggctt gcgctattcc caaaattgtg
      661 ggtattattt tcatatatca gtatctgcaa tttcggttca actttcttat caagaaagag
      721 gattaatttc ttaacaaaaa ccttctttgt tttataacgc tgatttttgt attatttggc
      781 gaccaaggtt ataagtgatc caatccaaat gtcataactt cagttgatcc ctgcggaaaa
      841 tataaatcag attatggtta tataaaaaaa aagagaaatt atttgagttc attgaaactt
      901 caacgttatt tgactaaaac ttgttaattt acatgtctgc aagattaatc cagcatatta
      961 aataaattag ttgacttatg tcaaacaaat aatacattct tgatattatt agaaaaaaaa
     1021 aagaaaattg aataacttgt gagaaatgat attgttatta ttttagatta tgtcctttga
     1081 aatacaataa catgctaagg cacttactac ttaagccttt attactttaa tgtgctaaca
     1141 atcttctgta tccttagacg ttaaattttc ccttattaaa aaataaaaaa ataaaaaaaa
     1201 aatataggaa atgaaacaac cgctgatgac ttattctgtc ttttatggcg agatctgttt
     1261 agcttgcctc gtccccgccg ggtcacccgg ccagcgacat ggaggcccag aataccctcc
     1321 ttgacagtct tgacgtgcgc agctcagggg catgatgtga ctgtcgcccg tacatttagc
     1381 ccatacatcc ccatgtataa tcatttgcat ccatacattt tgatggccgc acggcgcgaa
     1441 gcaaaaatta cggctcctcg ctgcggacct gcgagcaggg aaacgctccc ctcacagacg
     1501 cgttgaattg tccccacgcc gcgcccctgt agagaaatat aaaaggttag gatttgccac
     1561 tgaggttctt ctttcatata cttcctttta aaatcttgct aggatacagt tctcacatca
     1621 catccgaaca taaacaacca tgggtaaaaa gcctgaactc accgcgacgt ctgtcgagaa
     1681 gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg agggcgaaga
     1741 atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg taaatagctg
     1801 cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc
     1861 gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt gcatctcccg
     1921 ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg ctgttctgca
     1981 gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga cgagcgggtt
     2041 cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt tcatatgcgc
     2101 gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg tcagtgcgtc
     2161 cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg aagtccggca
     2221 cctcatgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc gcataacagc
     2281 ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt
     2341 cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg agcggaggca
     2401 tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg gtcttgacca
     2461 actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc agggtcgatg
     2521 cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg cccgcagaag
     2581 cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa accgacgccc
     2641 cagcactcgt ccgagggcaa aggaataatc agtactgaca gtaaaaagat tcttgttttc
     2701 aagaacttgt catttgtata gtttttttat attgtagttg ttctatttta atcaaatgtt
     2761 agcgtgattt atattttttt tcgcctcgac atcatctgcc cagatgcgaa gttaagtgcg
     2821 cagaaagtaa tatcatgcgt caatcgtatg tgaatgctgg tcgctatact gctgtcgatt
     2881 cgatactaac gccgccatcc agtgtttaaa cgagctcgaa ttcatcgatg atatcagatc
     2941 cactagtggc ctatgcggcc gcggatctgc cggtctccct atagtgagtc gtattaattt
     3001 cgataagcca ggttaacctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3061 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3121 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     3181 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3241 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3301 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3361 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3421 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3481 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3541 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3601 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3661 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     3721 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     3781 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     3841 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     3901 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     3961 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4021 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4081 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     4141 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     4201 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     4261 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     4321 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     4381 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     4441 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4501 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4561 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     4621 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     4681 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     4741 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     4801 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     4861 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     4921 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     4981 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     5041 ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa
     5101 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     5161 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
     5221 atgcggcatc agagcagatt gtactgagag tgcaccatat ggacatattg tcgttagaac
     5281 gcggctacaa ttaatacata accttatgta
//
