LOCUS       sequence-203644-        6024 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 6024)
  AUTHORS   Liu Y, Lee IJ, Sun M, Lower CA, Runge KW, Ma J, Wu JQ
  TITLE     Roles of the novel coiled-coil protein Rng10 in septum formation 
            during fission yeast cytokinesis.
  JOURNAL   Mol Biol Cell. 2016 Aug 15;27(16):2528-41. doi: 
            10.1091/mbc.E16-03-0156. Epub 2016 Jul 6.
  PUBMED    27385337
REFERENCE   2  (bases 1 to 6024)
  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: 
            "10.1091/mbc.E16-03-0156"; journalName: "Mol Biol Cell"; date: 
            "2016-08-15- 15"; volume: "27"; issue: "16"; pages: "2528-41"
COMMENT     Sequence Label: pFA6a-kanMX6-3nmt1-mEOS3.2
FEATURES             Location/Qualifiers
     source          1..6024
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      7..595
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     496..515
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     749..766
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     853..872
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        853..871
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     gene            complement(976..2332)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(976..1173)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1179..1988)
                     /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     1902..1921
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1989..2332)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        2377..3540
                     /label=nmt1 promoter
                     /note="wild-type nmt1 promoter from Schizosaccharomyces 
                     pombe, conferring strong thiamine-repressible expression"
     CDS             3567..4247
                     /codon_start=1
                     /product="improved monomeric variant of green-to-red 
                     photoswitchable fluorescent protein EosFP (Zhang et al., 
                     2012)"
                     /label=mEos3.2
                     /translation="MSAIKPDMKIKLRMEGNVNGHHFVIDGDGTGKPFEGKQSMDLEVK
                     EGGPLPFAFDILTTAFHYGNRVFAKYPDNIQDYFKQSFPKGYSWERSLTFEDGGICNAR
                     NDITMEGDTFYNKVRFYGTNFPANGPVMQKKTLKWEPSTEKMYVRDGVLTGDIEMALLL
                     EGNAHYRCDFRTTYKAKEKGVKLPGAHFVDHCIEILSHDKDYNKVKLYEHAVAHSGLPD
                     NARR"
     terminator      4268..4455
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4323..4347)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(4531..4549)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4532..4549)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4630..4649)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4749..4771
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4809..4827)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4895..4999
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5000..5068,5069..5860)
                     /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(5218..5237)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
       61 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
      121 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
      181 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
      241 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
      301 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
      361 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
      421 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
      481 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
      541 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
      601 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
      661 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
      721 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
      781 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca ggttaacctg
      841 gcttatcgaa attaatacga ctcactatag ggagaccggc agatccgcgg ccgcataggc
      901 cactagtgga tctgatatca tcgatgaatt cgagctcgtt taaactggat ggcggcgtta
      961 gtatcgaatc gacagcagta tagcgaccag cattcacata cgattgacgc atgatattac
     1021 tttctgcgca cttaacttcg catctgggca gatgatgtcg aggcgaaaaa aaatataaat
     1081 cacgctaaca tttgattaaa atagaacaac tacaatataa aaaaactata caaatgacaa
     1141 gttcttgaaa acaagaatct ttttattgtc agtactgatt agaaaaactc atcgagcatc
     1201 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     1261 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     1321 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     1381 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     1441 agcttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     1501 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     1561 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     1621 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     1681 gttttgccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     1741 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     1801 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     1861 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     1921 ccatataaat cagcatccat gttggaattt aatcgcggcc tcgaaacgtg agtcttttcc
     1981 ttacccatgg ttgtttatgt tcggatgtga tgtgagaact gtatcctagc aagattttaa
     2041 aaggaagtat atgaaagaag aacctcagtg gcaaatccta accttttata tttctctaca
     2101 ggggcgcggc gtggggacaa ttcaacgcgt ctgtgagggg agcgtttccc tgctcgcagg
     2161 tctgcagcga ggagccgtaa tttttgcttc gcgccgtgcg gccatcaaaa tgtatggatg
     2221 caaatgatta tacatgggga tgtatgggct aaatgtacgg gcgacagtca catcatgccc
     2281 ctgagctgcg cacgtcaaga ctgtcaagga gggtattctg ggcctccatg tcgctggccg
     2341 ggtgacccgg cggggacgag gcaagctaaa cagatctcgc cataaaagac agaataagtc
     2401 atcagcggtt gtttcatttc ctatattttt tttttatttt tttatttttt aataagggaa
     2461 aatttaacgt ctaaggatac agaagattgt tagcacatta aagtaataaa ggcttaagta
     2521 gtaagtgcct tagcatgtta ttgtatttca aaggacataa tctaaaataa taacaatatc
     2581 atttctcaca agttattcaa ttttcttttt ttttctaata atatcaagaa tgtattattt
     2641 gtttgacata agtcaactaa tttatttaat atgctggatt aatcttgcag acatgtaaat
     2701 taacaagttt tagtcaaata acgttgaagt ttcaatgaac tcaaataatt tctctttttt
     2761 tttatataac catatgtcta 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 tgaagatata taaaggaaga ggaatcctgg catatcatca attgaataag
     3481 ttgaattaat tatttcaatc tcattctcac tttctgactt atagtcgctt tgttaaatca
     3541 tgtctttaat taacggaggt ggaggtatga gtgcgattaa gccagacatg aagatcaaac
     3601 tccgtatgga aggcaacgta aacgggcacc actttgtgat cgacggagat ggtacaggca
     3661 agccttttga gggaaaacag agtatggatc ttgaagtcaa agagggcgga cctctgcctt
     3721 ttgcctttga tatcctgacc actgcattcc attacggcaa cagggtattc gccaaatatc
     3781 cagacaacat acaagactat tttaagcagt cgtttcctaa ggggtattcg tgggaacgaa
     3841 gcttgacttt cgaagacggg ggcatttgca acgccagaaa cgacataaca atggaagggg
     3901 acactttcta taataaagtt cgattttatg gtaccaactt tcccgccaat ggtccagtta
     3961 tgcagaagaa gacgctgaaa tgggagccct ccactgagaa aatgtatgtg cgtgatggag
     4021 tgctgacggg tgatattgag atggctttgt tgcttgaagg aaatgcccat taccgatgtg
     4081 acttcagaac tacttacaaa gctaaggaga agggtgtcaa gttaccaggc gcccactttg
     4141 tggaccactg cattgagatt ttaagccatg acaaagatta caacaaggtt aagctgtatg
     4201 agcatgctgt tgctcattct ggattgcctg acaatgccag acgataaggc gcgccacttc
     4261 taaataagcg aatttcttat gatttatgat ttttattatt aaataagtta taaaaaaaat
     4321 aagtgtatac aaattttaaa gtgactctta ggttttaaaa cgaaaattct tattcttgag
     4381 taactctttc ctgtaggtca ggttgctttc tcaggtatag tatgaggtcg ctcttattga
     4441 ccacacctct accggcagat ccgctaggga taacagggta atatagatcc gtcgacctgc
     4501 agcgtacgaa gcttcagctg gcggccgcgt tctatagtgt cacctaaatc gtatgtgtat
     4561 gatacataag gttatgtatt aattgtagcc gcgttctaac gacaatatgt ccatatggtg
     4621 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     4681 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4741 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4801 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     4861 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     4921 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     4981 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     5041 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     5101 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     5161 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     5221 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     5281 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     5341 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     5401 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     5461 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     5521 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     5581 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     5641 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     5701 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     5761 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     5821 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     5881 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     5941 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     6001 agaccccgta gaaaagatca aagg
//
