LOCUS       sequence-203270-        5307 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 5307)
  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 5307)
  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-hphMX6-81nmt1
FEATURES             Location/Qualifiers
     source          1..5307
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        6..24
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     6..23
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        complement(89..1240)
                     /label=nmt1 P81 promoter
                     /note="mutant nmt1 promoter from Schizosaccharomyces pombe,
                     conferring weak thiamine-repressible expression"
     gene            1285..2860
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        1285..1628
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1629..2657
                     /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      2663..2860
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2966..2985)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2967..2985)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3072..3089)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3243..3831)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3323..3342)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4002..4793,4794..4862))
                     /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     4625..4644
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4863..4967)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5035..5053
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5091..5113)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5213..5232
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atacgattta ggtgacacta tagaacgcgg ccgccagctg aagcttcgta cgctgcaggt
       61 cgacggatcc ccgggttaat taaagacatg atttaacaaa gcgactataa gtcagaaagt
      121 gagaatgaga ttgaaataat taattcaact tattcaattg atgatatgcc aggattcctc
      181 ttccatcttc aatgtttcga ttaacaggtc cattatccgg tttttagttt cgctgcacat
      241 tgccgaatga caatcggatt cctttaagct ttgcttttgc cctgttttct ctccccagta
      301 atcgttgggt gactgaacca tccagtagcc gagagttacg tttttcctga tttaataaat
      361 atccatatct atttgtctcg tggtgtttta cagcaactaa ccgaaattac tattttgcat
      421 catccaacca taaagagaca acaataattc tagaaaacat ataacaagtg tttttttcta
      481 tttacgtctc atacgcaaag tccactttac catgttttgg aaacttgtac ccaaccattc
      541 atttttcgat tttgtaatga ggctcttttc ttctaacaat cattgaggtc attatcatgt
      601 gtcctcacct attgtttaaa ttgaggcttg cgctattccc aaaattgtgg gtattatttt
      661 catatatcag tatctgcaat ttcggttcaa ctttcttatc aagaaagagg attaatttct
      721 taacaaaaac cttctttgtt ttataacgct gatttttgta ttatttggcg accaaggtta
      781 taagtgatcc aatccaaatg tcataacttc agttgatccc tgcggaaaat ataaatcaga
      841 ttatggttat ataaaaaaaa agagaaatta tttgagttca ttgaaacttc aacgttattt
      901 gactaaaact tgttaattta catgtctgca agattaatcc agcatattaa ataaattagt
      961 tgacttatgt caaacaaata atacattctt gatattatta gaaaaaaaaa agaaaattga
     1021 ataacttgtg agaaatgata ttgttattat tttagattat gtcctttgaa atacaataac
     1081 atgctaaggc acttactact taagccttta ttactttaat gtgctaacaa tcttctgtat
     1141 ccttagacgt taaattttcc cttattaaaa aataaaaaaa taaaaaaaaa atataggaaa
     1201 tgaaacaacc gctgatgact tattctgtct tttatggcga gatctgttta gcttgcctcg
     1261 tccccgccgg gtcacccggc cagcgacatg gaggcccaga ataccctcct tgacagtctt
     1321 gacgtgcgca gctcaggggc atgatgtgac tgtcgcccgt acatttagcc catacatccc
     1381 catgtataat catttgcatc catacatttt gatggccgca cggcgcgaag caaaaattac
     1441 ggctcctcgc tgcggacctg cgagcaggga aacgctcccc tcacagacgc gttgaattgt
     1501 ccccacgccg cgcccctgta gagaaatata aaaggttagg atttgccact gaggttcttc
     1561 tttcatatac ttccttttaa aatcttgcta ggatacagtt ctcacatcac atccgaacat
     1621 aaacaaccat gggtaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg
     1681 aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt
     1741 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt
     1801 tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag
     1861 tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg
     1921 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg
     1981 aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg
     2041 gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc
     2101 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg
     2161 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcatgcacg
     2221 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact
     2281 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc
     2341 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg
     2401 caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga
     2461 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg
     2521 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct
     2581 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc
     2641 cgagggcaaa ggaataatca gtactgacag taaaaagatt cttgttttca agaacttgtc
     2701 atttgtatag tttttttata ttgtagttgt tctattttaa tcaaatgtta gcgtgattta
     2761 tatttttttt cgcctcgaca tcatctgccc agatgcgaag ttaagtgcgc agaaagtaat
     2821 atcatgcgtc aatcgtatgt gaatgctggt cgctatactg ctgtcgattc gatactaacg
     2881 ccgccatcca gtgtttaaac gagctcgaat tcatcgatga tatcagatcc actagtggcc
     2941 tatgcggccg cggatctgcc ggtctcccta tagtgagtcg tattaatttc gataagccag
     3001 gttaacctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     3061 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3121 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3181 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3241 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3301 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3361 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3421 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     3481 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3541 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     3601 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     3661 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     3721 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     3781 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     3841 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     3901 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3961 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4021 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4081 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     4141 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4201 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4261 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     4321 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     4381 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     4441 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     4501 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4561 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     4621 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4681 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4741 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4801 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4861 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4921 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     4981 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     5041 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     5101 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     5161 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
     5221 gagcagattg tactgagagt gcaccatatg gacatattgt cgttagaacg cggctacaat
     5281 taatacataa ccttatgtat catacac
//
