LOCUS       sequence-203643-        6033 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 6033)
  AUTHORS   Laporte D, Ojkic N, Vavylonis D, Wu JQ
  TITLE     alpha-Actinin and fimbrin cooperate with myosin II to organize 
            actomyosin bundles during contractile-ring assembly.
  JOURNAL   Mol Biol Cell. 2012 Aug;23(16):3094-110. doi: 
            10.1091/mbc.E12-02-0123. Epub 2012 Jun 27.
  PUBMED    22740629
REFERENCE   2  (bases 1 to 6033)
  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"; doi: 
            "10.1091/mbc.E12-02-0123"; journalName: "Mol Biol Cell"; date: 
            "2012-08"; volume: "23"; issue: "16"; pages: "3094-110"
COMMENT     Sequence Label: pFA6a-kanMX6-P81nmt1-mEGFP
FEATURES             Location/Qualifiers
     source          1..6033
                     /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..3541
                     /label=nmt1 P81 promoter
                     /note="mutant nmt1 promoter from Schizosaccharomyces pombe,
                     conferring weak thiamine-repressible expression"
     CDS             3556..4266
                     /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
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLSPDNHYLSTQSKLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             3556..4266
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLSPDNHYLSTQSKLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(3583..3611)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4206..4226
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      4290..4477
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4345..4369)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(4553..4571)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4554..4571)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4652..4671)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4771..4793
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4831..4849)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4917..5021
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5022..5090,5091..5882)
                     /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(5240..5259)
                     /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 aagatggaag aggaatcctg gcatatcatc aattgaataa
     3481 gttgaattaa ttatttcaat ctcattctca ctttctgact tatagtcgct ttgttaaatc
     3541 atgtctttaa ttaacagtaa aggagaagaa cttttcactg gagttgtccc aattcttgtt
     3601 gaattagatg gtgacgttaa tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat
     3661 gcaacatacg gaaaacttac ccttaaattt atttgcacta ctggaaaact acctgttcca
     3721 tggccaacac ttgtcactac tctgacttat ggtgttcaat gcttttcaag atacccagat
     3781 catatgaaac agcatgactt tttcaagagt gccatgcccg aaggttatgt acaggaaaga
     3841 actatatttt tcaaagatga cgggaactac aagacacgtg ctgaagtcaa gtttgaaggt
     3901 gatacccttg ttaatagaat cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt
     3961 cttggacaca aattggaata caactataac tcacacaatg tatacatcat ggcagacaaa
     4021 caaaagaatg gaatcaaagt taacttcaaa attagacaca acattgaaga tggaagcgtt
     4081 caactagcag accattatca acaaaatact ccaattggcg atggccctgt cctttcacca
     4141 gacaaccatt acctgtccac acaatctaag ctttcgaaag atcccaacga aaagagagac
     4201 cacatggtcc ttcttgagtt tgtaacagct gctgggatta cacatggcat ggatgaacta
     4261 tacaaatagg gcgcgccact tctaaataag cgaatttctt atgatttatg atttttatta
     4321 ttaaataagt tataaaaaaa ataagtgtat acaaatttta aagtgactct taggttttaa
     4381 aacgaaaatt cttattcttg agtaactctt tcctgtaggt caggttgctt tctcaggtat
     4441 agtatgaggt cgctcttatt gaccacacct ctaccggcag atccgctagg gataacaggg
     4501 taatatagat ccgtcgacct gcagcgtacg aagcttcagc tggcggccgc gttctatagt
     4561 gtcacctaaa tcgtatgtgt atgatacata aggttatgta ttaattgtag ccgcgttcta
     4621 acgacaatat gtccatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     4681 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     4741 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     4801 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     4861 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     4921 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     4981 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     5041 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     5101 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     5161 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     5221 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     5281 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     5341 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     5401 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     5461 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     5521 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     5581 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     5641 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     5701 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     5761 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     5821 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     5881 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     5941 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     6001 agttttcgtt ccactgagcg tcagaccccg tag
//
