LOCUS       sequence-203266-        5319 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 5319)
  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 5319)
  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-hphMX6-3nmt1
FEATURES             Location/Qualifiers
     source          1..5319
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        18..36
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     18..35
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        complement(101..1264)
                     /label=nmt1 promoter
                     /note="wild-type nmt1 promoter from Schizosaccharomyces 
                     pombe, conferring strong thiamine-repressible expression"
     gene            1309..2884
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        1309..1652
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1653..2681
                     /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      2687..2884
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2990..3009)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2991..3009)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3096..3113)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3267..3855)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3347..3366)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4026..4817,4818..4886))
                     /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     4649..4668
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4887..4991)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5059..5077
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5115..5137)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5237..5256
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgtatcatac acatacgatt taggtgacac tatagaacgc ggccgccagc tgaagcttcg
       61 tacgctgcag gtcgacggat ccccgggtta attaaagaca tgatttaaca aagcgactat
      121 aagtcagaaa gtgagaatga gattgaaata attaattcaa cttattcaat tgatgatatg
      181 ccaggattcc tcttccttta tatatcttca atgtttcgat taacaggtcc attatccggt
      241 ttttagtttc gctgcacatt gccgaatgac aatcggattc ctttaagctt tgcttttgcc
      301 ctgttttctc tccccagtaa tcgttgggtg actgaaccat ccagtagccg agagttacgt
      361 ttttcctgat ttaataaata tccatatcta tttgtctcgt ggtgttttac agcaactaac
      421 cgaaattact attttgcatc atccaaccat aaagagacaa caataattct agaaaacata
      481 taacaagtgt ttttttctat ttacgtctca tacgcaaagt ccactttacc atgttttgga
      541 aacttgtacc caaccattca tttttcgatt ttgtaatgag gctcttttct tctaacaatc
      601 attgaggtca ttatcatgtg tcctcaccta ttgtttaaat tgaggcttgc gctattccca
      661 aaattgtggg tattattttc atatatcagt atctgcaatt tcggttcaac tttcttatca
      721 agaaagagga ttaatttctt aacaaaaacc ttctttgttt tataacgctg atttttgtat
      781 tatttggcga ccaaggttat aagtgatcca atccaaatgt cataacttca gttgatccct
      841 gcggaaaata taaatcagat tagacatatg gttatataaa aaaaaagaga aattatttga
      901 gttcattgaa acttcaacgt tatttgacta aaacttgtta atttacatgt ctgcaagatt
      961 aatccagcat attaaataaa ttagttgact tatgtcaaac aaataataca ttcttgatat
     1021 tattagaaaa aaaaagaaaa ttgaataact tgtgagaaat gatattgtta ttattttaga
     1081 ttatgtcctt tgaaatacaa taacatgcta aggcacttac tacttaagcc tttattactt
     1141 taatgtgcta acaatcttct gtatccttag acgttaaatt ttcccttatt aaaaaataaa
     1201 aaaataaaaa aaaaatatag gaaatgaaac aaccgctgat gacttattct gtcttttatg
     1261 gcgagatctg tttagcttgc ctcgtccccg ccgggtcacc cggccagcga catggaggcc
     1321 cagaataccc tccttgacag tcttgacgtg cgcagctcag gggcatgatg tgactgtcgc
     1381 ccgtacattt agcccataca tccccatgta taatcatttg catccataca ttttgatggc
     1441 cgcacggcgc gaagcaaaaa ttacggctcc tcgctgcgga cctgcgagca gggaaacgct
     1501 cccctcacag acgcgttgaa ttgtccccac gccgcgcccc tgtagagaaa tataaaaggt
     1561 taggatttgc cactgaggtt cttctttcat atacttcctt ttaaaatctt gctaggatac
     1621 agttctcaca tcacatccga acataaacaa ccatgggtaa aaagcctgaa ctcaccgcga
     1681 cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct
     1741 cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc
     1801 gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat
     1861 cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct
     1921 attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc
     1981 ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc
     2041 agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg
     2101 atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca
     2161 ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc
     2221 ccgaagtccg gcacctcatg cacgcggatt tcggctccaa caatgtcctg acggacaatg
     2281 gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg
     2341 tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact
     2401 tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca
     2461 ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg
     2521 cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa
     2581 tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg
     2641 gaaaccgacg ccccagcact cgtccgaggg caaaggaata atcagtactg acagtaaaaa
     2701 gattcttgtt ttcaagaact tgtcatttgt atagtttttt tatattgtag ttgttctatt
     2761 ttaatcaaat gttagcgtga tttatatttt ttttcgcctc gacatcatct gcccagatgc
     2821 gaagttaagt gcgcagaaag taatatcatg cgtcaatcgt atgtgaatgc tggtcgctat
     2881 actgctgtcg attcgatact aacgccgcca tccagtgttt aaacgagctc gaattcatcg
     2941 atgatatcag atccactagt ggcctatgcg gccgcggatc tgccggtctc cctatagtga
     3001 gtcgtattaa tttcgataag ccaggttaac ctgcattaat gaatcggcca acgcgcgggg
     3061 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     3121 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     3181 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     3241 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     3301 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     3361 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     3421 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     3481 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     3541 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     3601 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     3661 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     3721 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     3781 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     3841 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     3901 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     3961 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     4021 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     4081 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     4141 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     4201 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     4261 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     4321 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     4381 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     4441 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     4501 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     4561 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     4621 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     4681 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     4741 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     4801 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     4861 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     4921 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     4981 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     5041 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     5101 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     5161 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     5221 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatggacata
     5281 ttgtcgttag aacgcggcta caattaatac ataacctta
//
