LOCUS       sequence-203658-        6045 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 6045)
  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 6045)
  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-P3nmt1-mYFP
FEATURES             Location/Qualifiers
     source          1..6045
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      21..609
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     510..529
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     763..780
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     867..886
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        867..885
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     gene            complement(990..2346)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(990..1187)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1193..2002)
                     /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     1916..1935
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2003..2346)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        2391..3554
                     /label=nmt1 promoter
                     /note="wild-type nmt1 promoter from Schizosaccharomyces 
                     pombe, conferring strong thiamine-repressible expression"
     CDS             3569..4282
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(3596..3624)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4219..4239
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      4303..4490
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4358..4382)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(4566..4584)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4567..4584)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4665..4684)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4784..4806
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4844..4862)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4930..5034
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5035..5103,5104..5895)
                     /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(5253..5272)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
       61 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
      121 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
      181 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
      241 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
      301 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
      361 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
      421 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
      481 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
      541 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
      601 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
      661 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
      721 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
      781 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
      841 tgcaggttaa cctggcttat cgaaattaat acgactcact atagggagac cggcagatcc
      901 gcggccgcat aggccactag tggatctgat atcatcgatg aattcgagct cgtttaaact
      961 ggatggcggc gttagtatcg aatcgacagc agtatagcga ccagcattca catacgattg
     1021 acgcatgata ttactttctg cgcacttaac ttcgcatctg ggcagatgat gtcgaggcga
     1081 aaaaaaatat aaatcacgct aacatttgat taaaatagaa caactacaat ataaaaaaac
     1141 tatacaaatg acaagttctt gaaaacaaga atctttttat tgtcagtact gattagaaaa
     1201 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     1261 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     1321 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     1381 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     1441 gtgagaatgg caaaagctta tgcatttctt tccagacttg ttcaacaggc cagccattac
     1501 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     1561 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     1621 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     1681 atacctggaa tgctgttttg ccggggatcg cagtggtgag taaccatgca tcatcaggag
     1741 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     1801 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     1861 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     1921 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctcgaaa
     1981 cgtgagtctt ttccttaccc atggttgttt atgttcggat gtgatgtgag aactgtatcc
     2041 tagcaagatt ttaaaaggaa gtatatgaaa gaagaacctc agtggcaaat cctaaccttt
     2101 tatatttctc tacaggggcg cggcgtgggg acaattcaac gcgtctgtga ggggagcgtt
     2161 tccctgctcg caggtctgca gcgaggagcc gtaatttttg cttcgcgccg tgcggccatc
     2221 aaaatgtatg gatgcaaatg attatacatg gggatgtatg ggctaaatgt acgggcgaca
     2281 gtcacatcat gcccctgagc tgcgcacgtc aagactgtca aggagggtat tctgggcctc
     2341 catgtcgctg gccgggtgac ccggcgggga cgaggcaagc taaacagatc tcgccataaa
     2401 agacagaata agtcatcagc ggttgtttca tttcctatat ttttttttta tttttttatt
     2461 ttttaataag ggaaaattta acgtctaagg atacagaaga ttgttagcac attaaagtaa
     2521 taaaggctta agtagtaagt gccttagcat gttattgtat ttcaaaggac ataatctaaa
     2581 ataataacaa tatcatttct cacaagttat tcaattttct ttttttttct aataatatca
     2641 agaatgtatt atttgtttga cataagtcaa ctaatttatt taatatgctg gattaatctt
     2701 gcagacatgt aaattaacaa gttttagtca aataacgttg aagtttcaat gaactcaaat
     2761 aatttctctt tttttttata taaccatatg tctaatctga tttatatttt ccgcagggat
     2821 caactgaagt tatgacattt ggattggatc acttataacc ttggtcgcca aataatacaa
     2881 aaatcagcgt tataaaacaa agaaggtttt tgttaagaaa ttaatcctct ttcttgataa
     2941 gaaagttgaa ccgaaattgc agatactgat atatgaaaat aatacccaca attttgggaa
     3001 tagcgcaagc ctcaatttaa acaataggtg aggacacatg ataatgacct caatgattgt
     3061 tagaagaaaa gagcctcatt acaaaatcga aaaatgaatg gttgggtaca agtttccaaa
     3121 acatggtaaa gtggactttg cgtatgagac gtaaatagaa aaaaacactt gttatatgtt
     3181 ttctagaatt attgttgtct ctttatggtt ggatgatgca aaatagtaat ttcggttagt
     3241 tgctgtaaaa caccacgaga caaatagata tggatattta ttaaatcagg aaaaacgtaa
     3301 ctctcggcta ctggatggtt cagtcaccca acgattactg gggagagaaa acagggcaaa
     3361 agcaaagctt aaaggaatcc gattgtcatt cggcaatgtg cagcgaaact aaaaaccgga
     3421 taatggacct gttaatcgaa acattgaaga tatataaagg aagaggaatc ctggcatatc
     3481 atcaattgaa taagttgaat taattatttc aatctcattc tcactttctg acttatagtc
     3541 gctttgttaa atcatgtctt taattaacag taaaggagaa gaacttttca ctggagttgt
     3601 cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg tcagtggaga
     3661 gggtgaaggt gatgcaacat acggaaaact tacccttaaa tttatttgca ctactggaaa
     3721 actacctgtt ccatggccaa cacttgtcac tactttcggt tatggtctaa aatgctttgc
     3781 tagataccca gatcatatga aacggcatga ctttttcaag agtgccatgc ccgaaggtta
     3841 tgtacaggaa agaactatat ttttcaaaga tgacgggaac tacaagacac gtgctgaagt
     3901 caagtttgaa ggtgataccc ttgttaatag aatcgagtta aaaggtattg attttaaaga
     3961 agatggaaac attcttggac acaaattgga atacaactat aactcacaca atgtatacat
     4021 catggcagac aaacaaaaga atggaatcaa agttaacttc aaaattagac acaacattga
     4081 agatggaagc gttcaactag cagaccatta tcaacaaaat actccaattg gcgatggccc
     4141 tgtcctttta ccagacaacc attacctgtc ctatcaatct aagctttcga aagatcccaa
     4201 cgaaaagaga gaccacatgg tccttcttga gtttgtaaca gctgctggga ttacacatgg
     4261 catggatgaa ctatacaaat agggcgcgcc acttctaaat aagcgaattt cttatgattt
     4321 atgattttta ttattaaata agttataaaa aaaataagtg tatacaaatt ttaaagtgac
     4381 tcttaggttt taaaacgaaa attcttattc ttgagtaact ctttcctgta ggtcaggttg
     4441 ctttctcagg tatagtatga ggtcgctctt attgaccaca cctctaccgg cagatccgct
     4501 agggataaca gggtaatata gatccgtcga cctgcagcgt acgaagcttc agctggcggc
     4561 cgcgttctat agtgtcacct aaatcgtatg tgtatgatac ataaggttat gtattaattg
     4621 tagccgcgtt ctaacgacaa tatgtccata tggtgcactc tcagtacaat ctgctctgat
     4681 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4741 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4801 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     4861 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     4921 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     4981 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5041 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5101 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5161 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5221 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5281 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5341 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5401 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5461 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5521 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     5581 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5641 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     5701 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     5761 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     5821 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     5881 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     5941 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6001 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgta
//
