LOCUS       sequence-203251-        6036 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 6036)
  AUTHORS   Wang N, Lo Presti L, Zhu YH, Kang M, Wu Z, Martin SG, Wu JQ
  TITLE     The novel proteins Rng8 and Rng9 regulate the myosin-V Myo51 during 
            fission yeast cytokinesis.
  JOURNAL   J Cell Biol. 2014 May 12;205(3):357-75. doi: 10.1083/jcb.201308146. 
            Epub 2014 May 5.
  PUBMED    24798735
REFERENCE   2  (bases 1 to 6036)
  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: 
            "10.1083/jcb.201308146"; journalName: "J Cell Biol"; date: 
            "2014-05-12- 12"; volume: "205"; issue: "3"; pages: "357-75"
COMMENT     Sequence Label: pFA6a-kanMX6-P41nmt1-mECitrine
FEATURES             Location/Qualifiers
     source          1..6036
                     /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"
     terminator      complement(112..299)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     220..244
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(363..383)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     978..1006
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     promoter        complement(1048..2202)
                     /label=nmt1 P41 promoter
                     /note="mutant nmt1 promoter from Schizosaccharomyces pombe,
                     conferring medium strength thiamine-repressible expression"
     gene            2247..3603
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        2247..2590
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2591..3400
                     /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(2658..2677)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      3406..3603
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3707..3726)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3708..3726)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3813..3830)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3984..4572)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4064..4083)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4743..5534,5535..5603))
                     /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     5366..5385
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5604..5708)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5776..5794
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5832..5854)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5954..5973
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgtatcatac acatacgatt taggtgacac tatagaacgc ggccgccagc tgaagcttcg
       61 tacgctgcag gtcgacggat ctatattacc ctgttatccc tagcggatct gccggtagag
      121 gtgtggtcaa taagagcgac ctcatactat acctgagaaa gcaacctgac ctacaggaaa
      181 gagttactca agaataagaa ttttcgtttt aaaacctaag agtcacttta aaatttgtat
      241 acacttattt tttttataac ttatttaata ataaaaatca taaatcataa gaaattcgct
      301 tatttagaag tggcgcgccc tatttgtata gttcatccat gccatgtgta atcccagcag
      361 ctgttacaaa ctcaagaagg accatgtggt ctctcttttc gttgggatct ttcgaaagct
      421 tagattgata ggacaggtaa tggttgtctg gtaaaaggac agggccatcg ccaattggag
      481 tattttgttg ataatggtct gctagttgaa cgcttccatc ttcaatgttg tgtctaattt
      541 tgaagttaac tttgattcca ttcttttgtt tgtctgccat gatgtataca ttgtgtgagt
      601 tatagttgta ttccaatttg tgtccaagaa tgtttccatc ttctttaaaa tcaatacctt
      661 ttaactcgat tctattaaca agggtatcac cttcaaactt gacttcagca cgtgtcttgt
      721 agttcccgtc atctttgaaa aatatagttc tttcctgtac ataaccttcg ggcatggcac
      781 tcttgaaaaa gtcatgccgt ttcatatgat ctgggtatct agcaaagcac attagaccat
      841 aacccaaagt agtgacaagt gttggccatg gaacaggtag ttttccagta gtgcaaataa
      901 atttaagggt aagttttccg tatgttgcat caccttcacc ctctccactg acagaaaatt
      961 tgtgcccatt aacatcacca tctaattcaa caagaattgg gacaactcca gtgaaaagtt
     1021 cttctccttt actgttaatt aaagacatga tttaacaaag cgactataag tcagaaagtg
     1081 agaatgagat tgaaataatt aattcaactt attcaattga tgatatgcca ggattcctct
     1141 tcctttatct tcaatgtttc gattaacagg tccattatcc ggtttttagt ttcgctgcac
     1201 attgccgaat gacaatcgga ttcctttaag ctttgctttt gccctgtttt ctctccccag
     1261 taatcgttgg gtgactgaac catccagtag ccgagagtta cgtttttcct gatttaataa
     1321 atatccatat ctatttgtct cgtggtgttt tacagcaact aaccgaaatt actattttgc
     1381 atcatccaac cataaagaga caacaataat tctagaaaac atataacaag tgtttttttc
     1441 tatttacgtc tcatacgcaa agtccacttt accatgtttt ggaaacttgt acccaaccat
     1501 tcatttttcg attttgtaat gaggctcttt tcttctaaca atcattgagg tcattatcat
     1561 gtgtcctcac ctattgttta aattgaggct tgcgctattc ccaaaattgt gggtattatt
     1621 ttcatatatc agtatctgca atttcggttc aactttctta tcaagaaaga ggattaattt
     1681 cttaacaaaa accttctttg ttttataacg ctgatttttg tattatttgg cgaccaaggt
     1741 tataagtgat ccaatccaaa tgtcataact tcagttgatc cctgcggaaa atataaatca
     1801 gattatggtt atataaaaaa aaagagaaat tatttgagtt cattgaaact tcaacgttat
     1861 ttgactaaaa cttgttaatt tacatgtctg caagattaat ccagcatatt aaataaatta
     1921 gttgacttat gtcaaacaaa taatacattc ttgatattat tagaaaaaaa aaagaaaatt
     1981 gaataacttg tgagaaatga tattgttatt attttagatt atgtcctttg aaatacaata
     2041 acatgctaag gcacttacta cttaagcctt tattacttta atgtgctaac aatcttctgt
     2101 atccttagac gttaaatttt cccttattaa aaaataaaaa aataaaaaaa aaatatagga
     2161 aatgaaacaa ccgctgatga cttattctgt cttttatggc gagatctgtt tagcttgcct
     2221 cgtccccgcc gggtcacccg gccagcgaca tggaggccca gaataccctc cttgacagtc
     2281 ttgacgtgcg cagctcaggg gcatgatgtg actgtcgccc gtacatttag cccatacatc
     2341 cccatgtata atcatttgca tccatacatt ttgatggccg cacggcgcga agcaaaaatt
     2401 acggctcctc gctgcagacc tgcgagcagg gaaacgctcc cctcacagac gcgttgaatt
     2461 gtccccacgc cgcgcccctg tagagaaata taaaaggtta ggatttgcca ctgaggttct
     2521 tctttcatat acttcctttt aaaatcttgc taggatacag ttctcacatc acatccgaac
     2581 ataaacaacc atgggtaagg aaaagactca cgtttcgagg ccgcgattaa attccaacat
     2641 ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac
     2701 aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg
     2761 tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat
     2821 gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac
     2881 tgcgatcccc ggcaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa
     2941 tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg
     3001 tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg
     3061 tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg
     3121 gaaagaaatg cataagcttt tgccattctc accggattca gtcgtcactc atggtgattt
     3181 ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg
     3241 agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt
     3301 ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa
     3361 taaattgcag tttcatttga tgctcgatga gtttttctaa tcagtactga caataaaaag
     3421 attcttgttt tcaagaactt gtcatttgta tagttttttt atattgtagt tgttctattt
     3481 taatcaaatg ttagcgtgat ttatattttt tttcgcctcg acatcatctg cccagatgcg
     3541 aagttaagtg cgcagaaagt aatatcatgc gtcaatcgta tgtgaatgct ggtcgctata
     3601 ctgctgtcga ttcgatacta acgccgccat ccagtttaaa cgagctcgaa ttcatcgatg
     3661 atatcagatc cactagtggc ctatgcggcc gcggatctgc cggtctccct atagtgagtc
     3721 gtattaattt cgataagcca ggttaacctg cattaatgaa tcggccaacg cgcggggaga
     3781 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     3841 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     3901 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3961 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4021 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4081 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4141 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4201 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4261 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4321 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4381 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     4441 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4501 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4561 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4621 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4681 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     4741 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     4801 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     4861 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     4921 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     4981 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5041 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5101 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5161 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5221 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5281 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     5341 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     5401 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     5461 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     5521 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     5581 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     5641 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     5701 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     5761 acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat
     5821 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     5881 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     5941 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat ggacatattg
     6001 tcgttagaac gcggctacaa ttaatacata acctta
//
