LOCUS       sequence-203668-        6033 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 6033)
  AUTHORS   Wu JQ, Kuhn JR, Kovar DR, Pollard TD
  TITLE     Spatial and temporal pathway for assembly and constriction of the 
            contractile ring in fission yeast cytokinesis.
  JOURNAL   Dev Cell. 2003 Nov;5(5):723-34.
  PUBMED    14602073
REFERENCE   2  (bases 1 to 6033)
  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"; journalName: "Dev Cell.";
            date: "2003-11"; volume: "5(5)"; pages: "723-34"
COMMENT     Sequence Label: pFA6a-kanMX6-P81nmt1-mYFP
FEATURES             Location/Qualifiers
     source          1..6033
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      27..615
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     516..535
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     769..786
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     873..892
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        873..891
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     gene            complement(996..2352)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(996..1193)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1199..2008)
                     /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     1922..1941
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2009..2352)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        2397..3548
                     /label=nmt1 P81 promoter
                     /note="mutant nmt1 promoter from Schizosaccharomyces pombe,
                     conferring weak thiamine-repressible expression"
     CDS             3563..4276
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(3590..3618)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4213..4233
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      4297..4484
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4352..4376)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(4560..4578)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4561..4578)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4659..4678)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4778..4800
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4838..4856)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4924..5028
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5029..5097,5098..5889)
                     /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     complement(5247..5266)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
       61 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
      121 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
      181 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
      241 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
      301 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
      361 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
      421 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
      481 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
      541 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
      601 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
      661 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
      721 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
      781 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
      841 cattaatgca ggttaacctg gcttatcgaa attaatacga ctcactatag ggagaccggc
      901 agatccgcgg ccgcataggc cactagtgga tctgatatca tcgatgaatt cgagctcgtt
      961 taaactggat ggcggcgtta gtatcgaatc gacagcagta tagcgaccag cattcacata
     1021 cgattgacgc atgatattac tttctgcgca cttaacttcg catctgggca gatgatgtcg
     1081 aggcgaaaaa aaatataaat cacgctaaca tttgattaaa atagaacaac tacaatataa
     1141 aaaaactata caaatgacaa gttcttgaaa acaagaatct ttttattgtc agtactgatt
     1201 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
     1261 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     1321 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
     1381 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     1441 aatccggtga gaatggcaaa agcttatgca tttctttcca gacttgttca acaggccagc
     1501 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     1561 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     1621 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     1681 cttctaatac ctggaatgct gttttgccgg ggatcgcagt ggtgagtaac catgcatcat
     1741 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     1801 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     1861 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     1921 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     1981 tcgaaacgtg agtcttttcc ttacccatgg ttgtttatgt tcggatgtga tgtgagaact
     2041 gtatcctagc aagattttaa aaggaagtat atgaaagaag aacctcagtg gcaaatccta
     2101 accttttata tttctctaca ggggcgcggc gtggggacaa ttcaacgcgt ctgtgagggg
     2161 agcgtttccc tgctcgcagg tctgcagcga ggagccgtaa tttttgcttc gcgccgtgcg
     2221 gccatcaaaa tgtatggatg caaatgatta tacatgggga tgtatgggct aaatgtacgg
     2281 gcgacagtca catcatgccc ctgagctgcg cacgtcaaga ctgtcaagga gggtattctg
     2341 ggcctccatg tcgctggccg ggtgacccgg cggggacgag gcaagctaaa cagatctcgc
     2401 cataaaagac agaataagtc atcagcggtt gtttcatttc ctatattttt tttttatttt
     2461 tttatttttt aataagggaa aatttaacgt ctaaggatac agaagattgt tagcacatta
     2521 aagtaataaa ggcttaagta gtaagtgcct tagcatgtta ttgtatttca aaggacataa
     2581 tctaaaataa taacaatatc atttctcaca agttattcaa ttttcttttt tttttctaat
     2641 aatatcaaga atgtattatt tgtttgacat aagtcaacta atttatttaa tatgctggat
     2701 taatcttgca gacatgtaaa ttaacaagtt ttagtcaaat aacgttgaag tttcaatgaa
     2761 ctcaaataat ttctcttttt ttttatataa ccataatctg atttatattt tccgcaggga
     2821 tcaactgaag ttatgacatt tggattggat cacttataac cttggtcgcc aaataataca
     2881 aaaatcagcg ttataaaaca aagaaggttt ttgttaagaa attaatcctc tttcttgata
     2941 agaaagttga accgaaattg cagatactga tatatgaaaa taatacccac aattttggga
     3001 atagcgcaag cctcaattta aacaataggt gaggacacat gataatgacc tcaatgattg
     3061 ttagaagaaa agagcctcat tacaaaatcg aaaaatgaat ggttgggtac aagtttccaa
     3121 aacatggtaa agtggacttt gcgtatgaga cgtaaataga aaaaaacact tgttatatgt
     3181 tttctagaat tattgttgtc tctttatggt tggatgatgc aaaatagtaa tttcggttag
     3241 ttgctgtaaa acaccacgag acaaatagat atggatattt attaaatcag gaaaaacgta
     3301 actctcggct actggatggt tcagtcaccc aacgattact ggggagagaa aacagggcaa
     3361 aagcaaagct taaaggaatc cgattgtcat tcggcaatgt gcagcgaaac taaaaaccgg
     3421 ataatggacc tgttaatcga aacattgaag atggaagagg aatcctggca tatcatcaat
     3481 tgaataagtt gaattaatta tttcaatctc attctcactt tctgacttat agtcgctttg
     3541 ttaaatcatg tctttaatta acagtaaagg agaagaactt ttcactggag ttgtcccaat
     3601 tcttgttgaa ttagatggtg atgttaatgg gcacaaattt tctgtcagtg gagagggtga
     3661 aggtgatgca acatacggaa aacttaccct taaatttatt tgcactactg gaaaactacc
     3721 tgttccatgg ccaacacttg tcactacttt cggttatggt ctaaaatgct ttgctagata
     3781 cccagatcat atgaaacggc atgacttttt caagagtgcc atgcccgaag gttatgtaca
     3841 ggaaagaact atatttttca aagatgacgg gaactacaag acacgtgctg aagtcaagtt
     3901 tgaaggtgat acccttgtta atagaatcga gttaaaaggt attgatttta aagaagatgg
     3961 aaacattctt ggacacaaat tggaatacaa ctataactca cacaatgtat acatcatggc
     4021 agacaaacaa aagaatggaa tcaaagttaa cttcaaaatt agacacaaca ttgaagatgg
     4081 aagcgttcaa ctagcagacc attatcaaca aaatactcca attggcgatg gccctgtcct
     4141 tttaccagac aaccattacc tgtcctatca atctaagctt tcgaaagatc ccaacgaaaa
     4201 gagagaccac atggtccttc ttgagtttgt aacagctgct gggattacac atggcatgga
     4261 tgaactatac aaatagggcg cgccacttct aaataagcga atttcttatg atttatgatt
     4321 tttattatta aataagttat aaaaaaaata agtgtataca aattttaaag tgactcttag
     4381 gttttaaaac gaaaattctt attcttgagt aactctttcc tgtaggtcag gttgctttct
     4441 caggtatagt atgaggtcgc tcttattgac cacacctcta ccggcagatc cgctagggat
     4501 aacagggtaa tatagatccg tcgacctgca gcgtacgaag cttcagctgg cggccgcgtt
     4561 ctatagtgtc acctaaatcg tatgtgtatg atacataagg ttatgtatta attgtagccg
     4621 cgttctaacg acaatatgtc catatggtgc actctcagta caatctgctc tgatgccgca
     4681 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     4741 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     4801 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     4861 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     4921 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     4981 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     5041 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     5101 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     5161 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     5221 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     5281 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     5341 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5401 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5461 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5521 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     5581 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     5641 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     5701 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     5761 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     5821 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     5881 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     5941 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     6001 taacgtgagt tttcgttcca ctgagcgtca gac
//
