LOCUS       sequence-203662-        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   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 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"; journalName: "Dev Cell.";
            date: "2003-11"; volume: "5(5)"; pages: "723-34"
COMMENT     Sequence Label: pFA6a-kanMX6-P41nmt1-mYFP
FEATURES             Location/Qualifiers
     source          1..6036
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      20..608
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     509..528
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     762..779
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     866..885
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        866..884
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     gene            complement(989..2345)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(989..1186)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1192..2001)
                     /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     1915..1934
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2002..2345)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        2390..3544
                     /label=nmt1 P41 promoter
                     /note="mutant nmt1 promoter from Schizosaccharomyces pombe,
                     conferring medium strength thiamine-repressible expression"
     CDS             3559..4272
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(3586..3614)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4209..4229
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      4293..4480
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4348..4372)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(4556..4574)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4557..4574)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4655..4674)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4774..4796
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4834..4852)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4920..5024
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5025..5093,5094..5885)
                     /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(5243..5262)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
       61 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
      121 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
      181 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
      241 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
      301 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
      361 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
      421 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
      481 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
      541 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
      601 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
      661 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
      721 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
      781 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
      841 gcaggttaac ctggcttatc gaaattaata cgactcacta tagggagacc ggcagatccg
      901 cggccgcata ggccactagt ggatctgata tcatcgatga attcgagctc gtttaaactg
      961 gatggcggcg ttagtatcga atcgacagca gtatagcgac cagcattcac atacgattga
     1021 cgcatgatat tactttctgc gcacttaact tcgcatctgg gcagatgatg tcgaggcgaa
     1081 aaaaaatata aatcacgcta acatttgatt aaaatagaac aactacaata taaaaaaact
     1141 atacaaatga caagttcttg aaaacaagaa tctttttatt gtcagtactg attagaaaaa
     1201 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     1261 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     1321 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     1381 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     1441 tgagaatggc aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     1501 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     1561 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     1621 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     1681 tacctggaat gctgttttgc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     1741 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     1801 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     1861 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     1921 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctcgaaac
     1981 gtgagtcttt tccttaccca tggttgttta tgttcggatg tgatgtgaga actgtatcct
     2041 agcaagattt taaaaggaag tatatgaaag aagaacctca gtggcaaatc ctaacctttt
     2101 atatttctct acaggggcgc ggcgtgggga caattcaacg cgtctgtgag gggagcgttt
     2161 ccctgctcgc aggtctgcag cgaggagccg taatttttgc ttcgcgccgt gcggccatca
     2221 aaatgtatgg atgcaaatga ttatacatgg ggatgtatgg gctaaatgta cgggcgacag
     2281 tcacatcatg cccctgagct gcgcacgtca agactgtcaa ggagggtatt ctgggcctcc
     2341 atgtcgctgg ccgggtgacc cggcggggac gaggcaagct aaacagatct cgccataaaa
     2401 gacagaataa gtcatcagcg gttgtttcat ttcctatatt ttttttttat ttttttattt
     2461 tttaataagg gaaaatttaa cgtctaagga tacagaagat tgttagcaca ttaaagtaat
     2521 aaaggcttaa gtagtaagtg ccttagcatg ttattgtatt tcaaaggaca taatctaaaa
     2581 taataacaat atcatttctc acaagttatt caattttctt ttttttttct aataatatca
     2641 agaatgtatt atttgtttga cataagtcaa ctaatttatt taatatgctg gattaatctt
     2701 gcagacatgt aaattaacaa gttttagtca aataacgttg aagtttcaat gaactcaaat
     2761 aatttctctt tttttttata taaccataat ctgatttata ttttccgcag ggatcaactg
     2821 aagttatgac atttggattg gatcacttat aaccttggtc gccaaataat acaaaaatca
     2881 gcgttataaa acaaagaagg tttttgttaa gaaattaatc ctctttcttg ataagaaagt
     2941 tgaaccgaaa ttgcagatac tgatatatga aaataatacc cacaattttg ggaatagcgc
     3001 aagcctcaat ttaaacaata ggtgaggaca catgataatg acctcaatga ttgttagaag
     3061 aaaagagcct cattacaaaa tcgaaaaatg aatggttggg tacaagtttc caaaacatgg
     3121 taaagtggac tttgcgtatg agacgtaaat agaaaaaaac acttgttata tgttttctag
     3181 aattattgtt gtctctttat ggttggatga tgcaaaatag taatttcggt tagttgctgt
     3241 aaaacaccac gagacaaata gatatggata tttattaaat caggaaaaac gtaactctcg
     3301 gctactggat ggttcagtca cccaacgatt actggggaga gaaaacaggg caaaagcaaa
     3361 gcttaaagga atccgattgt cattcggcaa tgtgcagcga aactaaaaac cggataatgg
     3421 acctgttaat cgaaacattg aagataaagg aagaggaatc ctggcatatc atcaattgaa
     3481 taagttgaat taattatttc aatctcattc tcactttctg acttatagtc gctttgttaa
     3541 atcatgtctt taattaacag taaaggagaa gaacttttca ctggagttgt cccaattctt
     3601 gttgaattag atggtgatgt taatgggcac aaattttctg tcagtggaga gggtgaaggt
     3661 gatgcaacat acggaaaact tacccttaaa tttatttgca ctactggaaa actacctgtt
     3721 ccatggccaa cacttgtcac tactttcggt tatggtctaa aatgctttgc tagataccca
     3781 gatcatatga aacggcatga ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa
     3841 agaactatat ttttcaaaga tgacgggaac tacaagacac gtgctgaagt caagtttgaa
     3901 ggtgataccc ttgttaatag aatcgagtta aaaggtattg attttaaaga agatggaaac
     3961 attcttggac acaaattgga atacaactat aactcacaca atgtatacat catggcagac
     4021 aaacaaaaga atggaatcaa agttaacttc aaaattagac acaacattga agatggaagc
     4081 gttcaactag cagaccatta tcaacaaaat actccaattg gcgatggccc tgtcctttta
     4141 ccagacaacc attacctgtc ctatcaatct aagctttcga aagatcccaa cgaaaagaga
     4201 gaccacatgg tccttcttga gtttgtaaca gctgctggga ttacacatgg catggatgaa
     4261 ctatacaaat agggcgcgcc acttctaaat aagcgaattt cttatgattt atgattttta
     4321 ttattaaata agttataaaa aaaataagtg tatacaaatt ttaaagtgac tcttaggttt
     4381 taaaacgaaa attcttattc ttgagtaact ctttcctgta ggtcaggttg ctttctcagg
     4441 tatagtatga ggtcgctctt attgaccaca cctctaccgg cagatccgct agggataaca
     4501 gggtaatata gatccgtcga cctgcagcgt acgaagcttc agctggcggc cgcgttctat
     4561 agtgtcacct aaatcgtatg tgtatgatac ataaggttat gtattaattg tagccgcgtt
     4621 ctaacgacaa tatgtccata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     4681 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     4741 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     4801 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     4861 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     4921 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4981 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     5041 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     5101 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     5161 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     5221 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     5281 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     5341 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     5401 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     5461 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     5521 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     5581 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     5641 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5701 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     5761 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     5821 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     5881 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     5941 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     6001 gtgagttttc gttccactga gcgtcagacc ccgtag
//
