LOCUS       sequence-203660-        6036 bp    DNA     circular SYN 15-APR-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 Apr 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-mCFP
FEATURES             Location/Qualifiers
     source          1..6036
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..600
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     501..520
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     754..771
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     858..877
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        858..876
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     gene            complement(981..2337)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(981..1178)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1184..1993)
                     /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     1907..1926
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1994..2337)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        2382..3536
                     /label=nmt1 P41 promoter
                     /note="mutant nmt1 promoter from Schizosaccharomyces pombe,
                     conferring medium strength thiamine-repressible expression"
     CDS             3551..4261
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKAH
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(3578..3606)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4201..4221
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      4285..4472
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4340..4364)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(4548..4566)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4549..4566)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4647..4666)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4766..4788
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4826..4844)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4912..5016
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5017..5085,5086..5877)
                     /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(5235..5254)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
       61 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
      121 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
      181 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
      241 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
      301 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
      361 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
      421 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
      481 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
      541 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
      601 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
      661 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
      721 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      781 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcaggtta
      841 acctggctta tcgaaattaa tacgactcac tatagggaga ccggcagatc cgcggccgca
      901 taggccacta gtggatctga tatcatcgat gaattcgagc tcgtttaaac tggatggcgg
      961 cgttagtatc gaatcgacag cagtatagcg accagcattc acatacgatt gacgcatgat
     1021 attactttct gcgcacttaa cttcgcatct gggcagatga tgtcgaggcg aaaaaaaata
     1081 taaatcacgc taacatttga ttaaaataga acaactacaa tataaaaaaa ctatacaaat
     1141 gacaagttct tgaaaacaag aatcttttta ttgtcagtac tgattagaaa aactcatcga
     1201 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     1261 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     1321 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     1381 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     1441 gcaaaagctt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     1501 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     1561 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     1621 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     1681 atgctgtttt gccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     1741 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     1801 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     1861 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     1921 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctcgaa acgtgagtct
     1981 tttccttacc catggttgtt tatgttcgga tgtgatgtga gaactgtatc ctagcaagat
     2041 tttaaaagga agtatatgaa agaagaacct cagtggcaaa tcctaacctt ttatatttct
     2101 ctacaggggc gcggcgtggg gacaattcaa cgcgtctgtg aggggagcgt ttccctgctc
     2161 gcaggtctgc agcgaggagc cgtaattttt gcttcgcgcc gtgcggccat caaaatgtat
     2221 ggatgcaaat gattatacat ggggatgtat gggctaaatg tacgggcgac agtcacatca
     2281 tgcccctgag ctgcgcacgt caagactgtc aaggagggta ttctgggcct ccatgtcgct
     2341 ggccgggtga cccggcgggg acgaggcaag ctaaacagat ctcgccataa aagacagaat
     2401 aagtcatcag cggttgtttc atttcctata tttttttttt atttttttat tttttaataa
     2461 gggaaaattt aacgtctaag gatacagaag attgttagca cattaaagta ataaaggctt
     2521 aagtagtaag tgccttagca tgttattgta tttcaaagga cataatctaa aataataaca
     2581 atatcatttc tcacaagtta ttcaattttc tttttttttt ctaataatat caagaatgta
     2641 ttatttgttt gacataagtc aactaattta tttaatatgc tggattaatc ttgcagacat
     2701 gtaaattaac aagttttagt caaataacgt tgaagtttca atgaactcaa ataatttctc
     2761 ttttttttta tataaccata atctgattta tattttccgc agggatcaac tgaagttatg
     2821 acatttggat tggatcactt ataaccttgg tcgccaaata atacaaaaat cagcgttata
     2881 aaacaaagaa ggtttttgtt aagaaattaa tcctctttct tgataagaaa gttgaaccga
     2941 aattgcagat actgatatat gaaaataata cccacaattt tgggaatagc gcaagcctca
     3001 atttaaacaa taggtgagga cacatgataa tgacctcaat gattgttaga agaaaagagc
     3061 ctcattacaa aatcgaaaaa tgaatggttg ggtacaagtt tccaaaacat ggtaaagtgg
     3121 actttgcgta tgagacgtaa atagaaaaaa acacttgtta tatgttttct agaattattg
     3181 ttgtctcttt atggttggat gatgcaaaat agtaatttcg gttagttgct gtaaaacacc
     3241 acgagacaaa tagatatgga tatttattaa atcaggaaaa acgtaactct cggctactgg
     3301 atggttcagt cacccaacga ttactgggga gagaaaacag ggcaaaagca aagcttaaag
     3361 gaatccgatt gtcattcggc aatgtgcagc gaaactaaaa accggataat ggacctgtta
     3421 atcgaaacat tgaagataaa ggaagaggaa tcctggcata tcatcaattg aataagttga
     3481 attaattatt tcaatctcat tctcactttc tgacttatag tcgctttgtt aaatcatgtc
     3541 tttaattaac agtaaaggag aagaactttt cactggagtt gtcccaattc ttgttgaatt
     3601 agatggtgat gttaatgggc acaaattttc tgtcagtgga gagggtgaag gtgatgcaac
     3661 atacggaaaa cttaccctta aatttatttg cactactgga aaactacctg ttccatggcc
     3721 aacacttgtc actactttga cttggggtgt tcaatgcttt tcaagatacc cagatcatat
     3781 gaaacggcat gactttttca agagtgccat gcccgaaggt tatgtacagg aaagaactat
     3841 atttttcaaa gatgacggga actacaagac acgtgctgaa gtcaagtttg aaggtgatac
     3901 ccttgttaat agaatcgagt taaaaggtat tgattttaaa gaagatggaa acattcttgg
     3961 acacaaattg gaatacaact atatttcaca caatgtatac atcactgcag acaaacaaaa
     4021 gaatggaatc aaagctcatt tcaaaattag acacaacatt gaagatggaa gcgttcaact
     4081 agcagaccat tatcaacaaa atactccaat tggcgatggc cctgtccttt taccagacaa
     4141 ccattacctg tccacacaat ctaagctttc gaaagatccc aacgaaaaga gagaccacat
     4201 ggtccttctt gagtttgtaa cagctgctgg gattacacat ggcatggatg aactatacaa
     4261 atagggcgcg ccacttctaa ataagcgaat ttcttatgat ttatgatttt tattattaaa
     4321 taagttataa aaaaaataag tgtatacaaa ttttaaagtg actcttaggt tttaaaacga
     4381 aaattcttat tcttgagtaa ctctttcctg taggtcaggt tgctttctca ggtatagtat
     4441 gaggtcgctc ttattgacca cacctctacc ggcagatccg ctagggataa cagggtaata
     4501 tagatccgtc gacctgcagc gtacgaagct tcagctggcg gccgcgttct atagtgtcac
     4561 ctaaatcgta tgtgtatgat acataaggtt atgtattaat tgtagccgcg ttctaacgac
     4621 aatatgtcca tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     4681 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     4741 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     4801 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     4861 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     4921 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     4981 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5041 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5101 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5161 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     5221 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     5281 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     5341 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     5401 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5461 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     5521 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     5581 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     5641 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     5701 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     5761 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     5821 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     5881 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     5941 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6001 tcgttccact gagcgtcaga ccccgtagaa aagatc
//
