LOCUS       sequence-204149-        6367 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 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 Dec 15, 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-3YFP-kanMX6
FEATURES             Location/Qualifiers
     source          1..6367
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        16..34
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     16..33
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             116..829
                     /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="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             116..829
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             836..1549
                     /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="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             836..1549
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             1556..2272
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      2314..2501
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2369..2393)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            2576..3932
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        2576..2919
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2920..3729
                     /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(2987..3006)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      3735..3932
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(4036..4055)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4037..4055)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4142..4159)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4313..4901)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4393..4412)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5072..5863,5864..5932))
                     /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     5695..5714
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5933..6037)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6105..6123
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6161..6183)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6283..6302
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tatcatacac atacgattta ggtgacacta tagaacgcgg ccgccagctg aagcttcgta
       61 cgctgcaggt cgacggatcc ccgggttaat taacggaaca aaagctggag gatccatgag
      121 taaaggagaa gaacttttca ctggagttgt cccaattctt gttgaattag atggtgatgt
      181 taatgggcac aaattttctg tcagtggaga gggtgaaggt gatgcaacat acggaaaact
      241 tacccttaaa tttatttgca ctactggaaa actacctgtt ccatggccaa cacttgtcac
      301 tactttcggt tatggtctaa aatgctttgc tagataccca gatcatatga aacggcatga
      361 ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa agaactatat ttttcaaaga
      421 tgacgggaac tacaagacac gtgctgaagt caagtttgaa ggtgataccc ttgttaatag
      481 aatcgagtta aaaggtattg attttaaaga agatggaaac attcttggac acaaattgga
      541 atacaactat aactcacaca atgtatacat catggcagac aaacaaaaga atggaatcaa
      601 agttaacttc aaaattagac acaacattga agatggaagc gttcaactag cagaccatta
      661 tcaacaaaat actccaattg gcgatggccc tgtcctttta ccagacaacc attacctgtc
      721 ctatcaatct gccctttcga aagatcccaa cgaaaagagg gaccacatgg tccttcttga
      781 gtttgtaaca gctgctggga ttacacatgg catggatgaa ctatacaaaa gatccatgag
      841 taaaggagaa gaacttttca ctggagttgt cccaattctt gttgaattag atggtgatgt
      901 taatgggcac aaattttctg tcagtggaga gggtgaaggt gatgcaacat acggaaaact
      961 tacccttaaa tttatttgca ctactggaaa actacctgtt ccatggccaa cacttgtcac
     1021 tactttcggt tatggtctaa aatgctttgc tagataccca gatcatatga aacggcatga
     1081 ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa agaactatat ttttcaaaga
     1141 tgacgggaac tacaagacac gtgctgaagt caagtttgaa ggtgataccc ttgttaatag
     1201 aatcgagtta aaaggtattg attttaaaga agatggaaac attcttggac acaaattgga
     1261 atacaactat aactcacaca atgtatacat catggcagac aaacaaaaga atggaatcaa
     1321 agttaacttc aaaattagac acaacattga agatggaagc gttcaactag cagaccatta
     1381 tcaacaaaat actccaattg gcgatggccc tgtcctttta ccagacaacc attacctgtc
     1441 ctatcaatct gccctttcga aagatcccaa cgaaaagagg gaccacatgg tccttcttga
     1501 gtttgtaaca gctgctggga ttacacatgg catggatgaa ctatacaaaa gatccatgag
     1561 taaaggagaa gaacttttca ctggagttgt cccaattctt gttgaattag atggtgatgt
     1621 taatgggcac aaattttctg tcagtggaga gggtgaaggt gatgcaacat acggaaaact
     1681 tacccttaaa tttatttgca ctactggaaa actacctgtt ccatggccaa cacttgtcac
     1741 tactttcggt tatggtctaa aatgctttgc tagataccca gatcatatga aacggcatga
     1801 ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa agaactatat ttttcaaaga
     1861 tgacgggaac tacaagacac gtgctgaagt caagtttgaa ggtgataccc ttgttaatag
     1921 aatcgagtta aaaggtattg attttaaaga agatggaaac attcttggac acaaattgga
     1981 atacaactat aactcacaca atgtatacat catggcagac aaacaaaaga atggaatcaa
     2041 agttaacttc aaaattagac acaacattga agatggaagc gttcaactag cagaccatta
     2101 tcaacaaaat actccaattg gcgatggccc tgtcctttta ccagacaacc attacctgtc
     2161 ctatcaatct gccctttcga aagatcccaa cgaaaagaga gaccacatgg tccttcttga
     2221 gtttgtaaca gctgctggga ttacacatgg catggatgaa ctatacaaat agagatctgt
     2281 attctttaat atcggcgcgc cacttctaaa taagcgaatt tcttatgatt tatgattttt
     2341 attattaaat aagttataaa aaaaataagt gtatacaaat tttaaagtga ctcttaggtt
     2401 ttaaaacgaa aattcttatt cttgagtaac tctttcctgt aggtcaggtt gctttctcag
     2461 gtatagtatg aggtcgctct tattgaccac acctctaccg gcagatccgc tagggataac
     2521 agggtaatat agatctgttt agcttgcctc gtccccgccg ggtcacccgg ccagcgacat
     2581 ggaggcccag aataccctcc ttgacagtct tgacgtgcgc agctcagggg catgatgtga
     2641 ctgtcgcccg tacatttagc ccatacatcc ccatgtataa tcatttgcat ccatacattt
     2701 tgatggccgc acggcgcgaa gcaaaaatta cggctcctcg ctgcagacct gcgagcaggg
     2761 aaacgctccc ctcacagacg cgttgaattg tccccacgcc gcgcccctgt agagaaatat
     2821 aaaaggttag gatttgccac tgaggttctt ctttcatata cttcctttta aaatcttgct
     2881 aggatacagt tctcacatca catccgaaca taaacaacca tgggtaagga aaagactcac
     2941 gtttcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     3001 cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg
     3061 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     3121 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     3181 actcctgatg atgcatggtt actcaccact gcgatccccg gcaaaacagc attccaggta
     3241 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     3301 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     3361 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     3421 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataagctttt gccattctca
     3481 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     3541 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     3601 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     3661 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     3721 tttttctaat cagtactgac aataaaaaga ttcttgtttt caagaacttg tcatttgtat
     3781 agttttttta tattgtagtt gttctatttt aatcaaatgt tagcgtgatt tatatttttt
     3841 ttcgcctcga catcatctgc ccagatgcga agttaagtgc gcagaaagta atatcatgcg
     3901 tcaatcgtat gtgaatgctg gtcgctatac tgctgtcgat tcgatactaa cgccgccatc
     3961 cagtttaaac gagctcgaat tcatcgatga tatcagatcc actagtggcc tatgcggccg
     4021 cggatctgcc ggtctcccta tagtgagtcg tattaatttc gataagccag gttaacctgc
     4081 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4141 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4201 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4261 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4321 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4381 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4441 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4501 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4561 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4621 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4681 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4741 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4801 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4861 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4921 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4981 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5041 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5101 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5161 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5221 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5281 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5341 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5401 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5461 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5521 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5581 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5641 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     5701 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5761 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5821 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     5881 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     5941 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6001 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6061 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     6121 ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     6181 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     6241 cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg
     6301 tactgagagt gcaccatatg gacatattgt cgttagaacg cggctacaat taatacataa
     6361 ccttatg
//
