LOCUS       sequence-203244-        4882 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 4882)
  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 4882)
  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- mECitrine-kanMX6
FEATURES             Location/Qualifiers
     source          1..4882
                     /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"
     primer_bind     complement(120..148)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     743..763
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      827..1014
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(882..906)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            1089..2445
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        1089..1432
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1433..2242
                     /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(1500..1519)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2248..2445
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2549..2568)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2550..2568)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2655..2672)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2826..3414)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2906..2925)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3585..4376,4377..4445))
                     /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     4208..4227
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4446..4550)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4618..4636
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4674..4696)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4796..4815
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tcatacacat acgatttagg tgacactata gaacgcggcc gccagctgaa gcttcgtacg
       61 ctgcaggtcg acggatcccc gggttaatta acagtaaagg agaagaactt ttcactggag
      121 ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg gcacaaattt tctgtcagtg
      181 gagagggtga aggtgatgca acatacggaa aacttaccct taaatttatt tgcactactg
      241 gaaaactacc tgttccatgg ccaacacttg tcactacttt gggttatggt ctaatgtgct
      301 ttgctagata cccagatcat atgaaacggc atgacttttt caagagtgcc atgcccgaag
      361 gttatgtaca ggaaagaact atatttttca aagatgacgg gaactacaag acacgtgctg
      421 aagtcaagtt tgaaggtgat acccttgtta atagaatcga gttaaaaggt attgatttta
      481 aagaagatgg aaacattctt ggacacaaat tggaatacaa ctataactca cacaatgtat
      541 acatcatggc agacaaacaa aagaatggaa tcaaagttaa cttcaaaatt agacacaaca
      601 ttgaagatgg aagcgttcaa ctagcagacc attatcaaca aaatactcca attggcgatg
      661 gccctgtcct tttaccagac aaccattacc tgtcctatca atctaagctt tcgaaagatc
      721 ccaacgaaaa gagagaccac atggtccttc ttgagtttgt aacagctgct gggattacac
      781 atggcatgga tgaactatac aaatagggcg cgccacttct aaataagcga atttcttatg
      841 atttatgatt tttattatta aataagttat aaaaaaaata agtgtataca aattttaaag
      901 tgactcttag gttttaaaac gaaaattctt attcttgagt aactctttcc tgtaggtcag
      961 gttgctttct caggtatagt atgaggtcgc tcttattgac cacacctcta ccggcagatc
     1021 cgctagggat aacagggtaa tatagatctg tttagcttgc ctcgtccccg ccgggtcacc
     1081 cggccagcga catggaggcc cagaataccc tccttgacag tcttgacgtg cgcagctcag
     1141 gggcatgatg tgactgtcgc ccgtacattt agcccataca tccccatgta taatcatttg
     1201 catccataca ttttgatggc cgcacggcgc gaagcaaaaa ttacggctcc tcgctgcaga
     1261 cctgcgagca gggaaacgct cccctcacag acgcgttgaa ttgtccccac gccgcgcccc
     1321 tgtagagaaa tataaaaggt taggatttgc cactgaggtt cttctttcat atacttcctt
     1381 ttaaaatctt gctaggatac agttctcaca tcacatccga acataaacaa ccatgggtaa
     1441 ggaaaagact cacgtttcga ggccgcgatt aaattccaac atggatgctg atttatatgg
     1501 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg
     1561 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     1621 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     1681 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggcaaaac
     1741 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     1801 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     1861 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     1921 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataagct
     1981 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     2041 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     2101 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     2161 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     2221 gatgctcgat gagtttttct aatcagtact gacaataaaa agattcttgt tttcaagaac
     2281 ttgtcatttg tatagttttt ttatattgta gttgttctat tttaatcaaa tgttagcgtg
     2341 atttatattt tttttcgcct cgacatcatc tgcccagatg cgaagttaag tgcgcagaaa
     2401 gtaatatcat gcgtcaatcg tatgtgaatg ctggtcgcta tactgctgtc gattcgatac
     2461 taacgccgcc atccagttta aacgagctcg aattcatcga tgatatcaga tccactagtg
     2521 gcctatgcgg ccgcggatct gccggtctcc ctatagtgag tcgtattaat ttcgataagc
     2581 caggttaacc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     2641 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     2701 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     2761 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     2821 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     2881 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     2941 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     3001 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     3061 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3121 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     3181 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3241 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     3301 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     3361 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     3421 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     3481 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     3541 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     3601 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     3661 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     3721 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     3781 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     3841 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     3901 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     3961 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     4021 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     4081 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     4141 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     4201 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     4261 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     4321 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     4381 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     4441 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     4501 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     4561 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     4621 aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga
     4681 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     4741 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca
     4801 tcagagcaga ttgtactgag agtgcaccat atggacatat tgtcgttaga acgcggctac
     4861 aattaataca taaccttatg ta
//
