LOCUS       sequence-204147-        6367 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Yeast gene targeting.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6367)
  AUTHORS   Wu JQ, Sirotkin V, Kovar DR, Lord M, Beltzner CC, Kuhn JR, Pollard 
            TD
  TITLE     Assembly of the cytokinetic contractile ring from a broad band of 
            nodes in fission yeast.
  JOURNAL   J Cell Biol. 2006 Jul 31;174(3):391-402. doi: 10.1083/jcb.200602032.
            Epub 2006 Jul 24.
  PUBMED    16864655
REFERENCE   2  (bases 1 to 6367)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1083/jcb.200602032"; journalName: "J Cell Biol"; date: 
            "2006-07-31- 31"; volume: "174"; issue: "3"; pages: "391-402"
COMMENT     Sequence Label: pFA6a-3GFP-kanMX6
FEATURES             Location/Qualifiers
     source          1..6367
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..42
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     24..41
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             124..837
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             844..1557
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             1564..2280
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      2322..2509
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2377..2401)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            2584..3940
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        2584..2927
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2928..3737
                     /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(2995..3014)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      3743..3940
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(4044..4063)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4045..4063)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4150..4167)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4321..4909)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4401..4420)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5080..5871,5872..5940))
                     /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     5703..5722
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5941..6045)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6113..6131
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6169..6191)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6291..6310
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 accttatgta tcatacacat acgatttagg tgacactata gaacgcggcc gccagctgaa
       61 gcttcgtacg ctgcaggtcg acggatcccc gggttaatta acggaacaaa agctggagga
      121 tccatgtcta aaggtgaaga attattcact ggtgttgtcc caattttggt tgaattagat
      181 ggtgatgtta atggtcacaa attttctgtc tccggtgaag gtgaaggtga tgctacttac
      241 ggtaaattga ccttaaaatt tatttgtact actggtaaat tgccagttcc atggccaacc
      301 ttagtcacta ctttcggtta tggtgttcaa tgttttgcga gatacccaga tcatatgaaa
      361 caacatgact ttttcaagtc tgccatgcca gaaggttatg ttcaagaaag aactattttt
      421 ttcaaagatg acggtaacta caagaccaga gctgaagtca agtttgaagg tgatacctta
      481 gttaatagaa tcgaattaaa aggtattgat tttaaagaag atggtaacat tttaggtcac
      541 aaattggaat acaactataa ctctcacaat gtttacatca tggctgacaa acaaaagaat
      601 ggtatcaaag ttaacttcaa aattagacac aacattgaag atggttctgt tcaattagct
      661 gaccattatc aacaaaatac tccaattggt gatggtccag tcttgttacc agacaaccat
      721 tacttatcca ctcaatctgc cttatccaaa gatccaaacg aaaagagaga ccacatggtc
      781 ttgttagaat ttgttactgc tgctggtatt acccatggta tggatgaatt gtacaaaaga
      841 tccatgtcta aaggtgaaga attattcact ggtgttgtcc caattttggt tgaattagat
      901 ggtgatgtta atggtcacaa attttctgtc tccggtgaag gtgaaggtga tgctacttac
      961 ggtaaattga ccttaaaatt tatttgtact actggtaaat tgccagttcc atggccaacc
     1021 ttagtcacta ctttcggtta tggtgttcaa tgttttgcga gatacccaga tcatatgaaa
     1081 caacatgact ttttcaagtc tgccatgcca gaaggttatg ttcaagaaag aactattttt
     1141 ttcaaagatg acggtaacta caagaccaga gctgaagtca agtttgaagg tgatacctta
     1201 gttaatagaa tcgaattaaa aggtattgat tttaaagaag atggtaacat tttaggtcac
     1261 aaattggaat acaactataa ctctcacaat gtttacatca tggctgacaa acaaaagaat
     1321 ggtatcaaag ttaacttcaa aattagacac aacattgaag atggttctgt tcaattagct
     1381 gaccattatc aacaaaatac tccaattggt gatggtccag tcttgttacc agacaaccat
     1441 tacttatcca ctcaatctgc cttatccaaa gatccaaacg aaaagagaga ccacatggtc
     1501 ttgttagaat ttgttactgc tgctggtatt acccatggta tggatgaatt gtacaaaaga
     1561 tccatgtcta aaggtgaaga attattcact ggtgttgtcc caattttggt tgaattagat
     1621 ggtgatgtta atggtcacaa attttctgtc tccggtgaag gtgaaggtga tgctacttac
     1681 ggtaaattga ccttaaaatt tatttgtact actggtaaat tgccagttcc atggccaacc
     1741 ttagtcacta ctttcggtta tggtgttcaa tgttttgcga gatacccaga tcatatgaaa
     1801 caacatgact ttttcaagtc tgccatgcca gaaggttatg ttcaagaaag aactattttt
     1861 ttcaaagatg acggtaacta caagaccaga gctgaagtca agtttgaagg tgatacctta
     1921 gttaatagaa tcgaattaaa aggtattgat tttaaagaag atggtaacat tttaggtcac
     1981 aaattggaat acaactataa ctctcacaat gtttacatca tggctgacaa acaaaagaat
     2041 ggtatcaaag ttaacttcaa aattagacac aacattgaag atggttctgt tcaattagct
     2101 gaccattatc aacaaaatac tccaattggt gatggtccag tcttgttacc agacaaccat
     2161 tacttatcca ctcaatctgc cttatccaaa gatccaaacg aaaagagaga ccacatggtc
     2221 ttgttagaat ttgttactgc tgctggtatt acccatggta tggatgaatt gtacaaataa
     2281 agatctgtat tctttaatat cggcgcgcca cttctaaata agcgaatttc ttatgattta
     2341 tgatttttat tattaaataa gttataaaaa aaataagtgt atacaaattt taaagtgact
     2401 cttaggtttt aaaacgaaaa ttcttattct tgagtaactc tttcctgtag gtcaggttgc
     2461 tttctcaggt atagtatgag gtcgctctta ttgaccacac ctctaccggc agatccgcta
     2521 gggataacag ggtaatatag atctgtttag cttgcctcgt ccccgccggg tcacccggcc
     2581 agcgacatgg aggcccagaa taccctcctt gacagtcttg acgtgcgcag ctcaggggca
     2641 tgatgtgact gtcgcccgta catttagccc atacatcccc atgtataatc atttgcatcc
     2701 atacattttg atggccgcac ggcgcgaagc aaaaattacg gctcctcgct gcagacctgc
     2761 gagcagggaa acgctcccct cacagacgcg ttgaattgtc cccacgccgc gcccctgtag
     2821 agaaatataa aaggttagga tttgccactg aggttcttct ttcatatact tccttttaaa
     2881 atcttgctag gatacagttc tcacatcaca tccgaacata aacaaccatg ggtaaggaaa
     2941 agactcacgt ttcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata
     3001 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg tatgggaagc
     3061 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag
     3121 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt
     3181 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggc aaaacagcat
     3241 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt
     3301 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat
     3361 ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg
     3421 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aagcttttgc
     3481 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg
     3541 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc
     3601 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc
     3661 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc
     3721 tcgatgagtt tttctaatca gtactgacaa taaaaagatt cttgttttca agaacttgtc
     3781 atttgtatag tttttttata ttgtagttgt tctattttaa tcaaatgtta gcgtgattta
     3841 tatttttttt cgcctcgaca tcatctgccc agatgcgaag ttaagtgcgc agaaagtaat
     3901 atcatgcgtc aatcgtatgt gaatgctggt cgctatactg ctgtcgattc gatactaacg
     3961 ccgccatcca gtttaaacga gctcgaattc atcgatgata tcagatccac tagtggccta
     4021 tgcggccgcg gatctgccgg tctccctata gtgagtcgta ttaatttcga taagccaggt
     4081 taacctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     4141 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     4201 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     4261 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     4321 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     4381 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     4441 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     4501 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     4561 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     4621 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     4681 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     4741 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     4801 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     4861 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     4921 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     4981 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5041 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     5101 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     5161 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     5221 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     5281 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     5341 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     5401 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     5461 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     5521 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     5581 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     5641 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     5701 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     5761 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     5821 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     5881 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     5941 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     6001 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     6061 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     6121 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     6181 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     6241 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
     6301 gcagattgta ctgagagtgc accatatgga catattgtcg ttagaacgcg gctacaatta
     6361 atacata
//
