LOCUS       sequence-203669-        4882 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 4882)
  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 4882)
  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- mCFP-kanMX6
FEATURES             Location/Qualifiers
     source          1..4882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(154..945,946..1014))
                     /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     777..796
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1015..1119)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1187..1205
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1243..1265)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1365..1384
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1465..1483
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1465..1482
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1544..2254
                     /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(1571..1599)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2194..2214
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2278..2465
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2333..2357)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            2540..3896
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        2540..2883
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2884..3693
                     /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(2951..2970)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      3699..3896
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(4000..4019)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4001..4019)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4106..4123)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4277..4865)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4357..4376)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
       61 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
      121 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
      181 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
      241 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
      301 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
      361 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
      421 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
      481 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
      541 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
      601 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
      661 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
      721 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
      781 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
      841 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
      901 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
      961 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     1021 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     1081 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     1141 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     1201 gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     1261 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     1321 cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat
     1381 tgtactgaga gtgcaccata tggacatatt gtcgttagaa cgcggctaca attaatacat
     1441 aaccttatgt atcatacaca tacgatttag gtgacactat agaacgcggc cgccagctga
     1501 agcttcgtac gctgcaggtc gacggatccc cgggttaatt aacagtaaag gagaagaact
     1561 tttcactgga gttgtcccaa ttcttgttga attagatggt gatgttaatg ggcacaaatt
     1621 ttctgtcagt ggagagggtg aaggtgatgc aacatacgga aaacttaccc ttaaatttat
     1681 ttgcactact ggaaaactac ctgttccatg gccaacactt gtcactactt tgacttgggg
     1741 tgttcaatgc ttttcaagat acccagatca tatgaaacgg catgactttt tcaagagtgc
     1801 catgcccgaa ggttatgtac aggaaagaac tatatttttc aaagatgacg ggaactacaa
     1861 gacacgtgct gaagtcaagt ttgaaggtga tacccttgtt aatagaatcg agttaaaagg
     1921 tattgatttt aaagaagatg gaaacattct tggacacaaa ttggaataca actatatttc
     1981 acacaatgta tacatcactg cagacaaaca aaagaatgga atcaaagctc atttcaaaat
     2041 tagacacaac attgaagatg gaagcgttca actagcagac cattatcaac aaaatactcc
     2101 aattggcgat ggccctgtcc ttttaccaga caaccattac ctgtccacac aatctaagct
     2161 ttcgaaagat cccaacgaaa agagagacca catggtcctt cttgagtttg taacagctgc
     2221 tgggattaca catggcatgg atgaactata caaatagggc gcgccacttc taaataagcg
     2281 aatttcttat gatttatgat ttttattatt aaataagtta taaaaaaaat aagtgtatac
     2341 aaattttaaa gtgactctta ggttttaaaa cgaaaattct tattcttgag taactctttc
     2401 ctgtaggtca ggttgctttc tcaggtatag tatgaggtcg ctcttattga ccacacctct
     2461 accggcagat ccgctaggga taacagggta atatagatct gtttagcttg cctcgtcccc
     2521 gccgggtcac ccggccagcg acatggaggc ccagaatacc ctccttgaca gtcttgacgt
     2581 gcgcagctca ggggcatgat gtgactgtcg cccgtacatt tagcccatac atccccatgt
     2641 ataatcattt gcatccatac attttgatgg ccgcacggcg cgaagcaaaa attacggctc
     2701 ctcgctgcag acctgcgagc agggaaacgc tcccctcaca gacgcgttga attgtcccca
     2761 cgccgcgccc ctgtagagaa atataaaagg ttaggatttg ccactgaggt tcttctttca
     2821 tatacttcct tttaaaatct tgctaggata cagttctcac atcacatccg aacataaaca
     2881 accatgggta aggaaaagac tcacgtttcg aggccgcgat taaattccaa catggatgct
     2941 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat
     3001 cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt
     3061 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt
     3121 ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc
     3181 cccggcaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt
     3241 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt
     3301 aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt
     3361 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa
     3421 atgcataagc ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt
     3481 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga
     3541 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct
     3601 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg
     3661 cagtttcatt tgatgctcga tgagtttttc taatcagtac tgacaataaa aagattcttg
     3721 ttttcaagaa cttgtcattt gtatagtttt tttatattgt agttgttcta ttttaatcaa
     3781 atgttagcgt gatttatatt ttttttcgcc tcgacatcat ctgcccagat gcgaagttaa
     3841 gtgcgcagaa agtaatatca tgcgtcaatc gtatgtgaat gctggtcgct atactgctgt
     3901 cgattcgata ctaacgccgc catccagttt aaacgagctc gaattcatcg atgatatcag
     3961 atccactagt ggcctatgcg gccgcggatc tgccggtctc cctatagtga gtcgtattaa
     4021 tttcgataag ccaggttaac ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4081 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4141 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4201 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4261 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4321 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4381 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4441 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4501 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4561 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4621 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4681 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     4741 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4801 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4861 ctcaagaaga tcctttgatc tt
//
