LOCUS       sequence-203646-        6051 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 6051)
  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 6051)
  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-mMaple3
FEATURES             Location/Qualifiers
     source          1..6051
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      16..604
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     505..524
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     758..775
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     862..881
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        862..880
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     gene            complement(985..2341)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(985..1182)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1188..1997)
                     /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     1911..1930
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1998..2341)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     promoter        2386..3549
                     /label=nmt1 promoter
                     /note="wild-type nmt1 promoter from Schizosaccharomyces 
                     pombe, conferring strong thiamine-repressible expression"
     terminator      4304..4491
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4359..4383)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(4567..4585)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4568..4585)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4666..4685)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4785..4807
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4845..4863)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4931..5035
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5036..5104,5105..5896)
                     /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(5254..5273)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
       61 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
      121 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
      181 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
      241 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
      301 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
      361 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
      421 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
      481 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
      541 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
      601 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
      661 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
      721 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
      781 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
      841 gttaacctgg cttatcgaaa ttaatacgac tcactatagg gagaccggca gatccgcggc
      901 cgcataggcc actagtggat ctgatatcat cgatgaattc gagctcgttt aaactggatg
      961 gcggcgttag tatcgaatcg acagcagtat agcgaccagc attcacatac gattgacgca
     1021 tgatattact ttctgcgcac ttaacttcgc atctgggcag atgatgtcga ggcgaaaaaa
     1081 aatataaatc acgctaacat ttgattaaaa tagaacaact acaatataaa aaaactatac
     1141 aaatgacaag ttcttgaaaa caagaatctt tttattgtca gtactgatta gaaaaactca
     1201 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
     1261 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
     1321 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
     1381 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     1441 aatggcaaaa gcttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     1501 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
     1561 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
     1621 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     1681 tggaatgctg ttttgccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     1741 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
     1801 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     1861 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     1921 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct cgaaacgtga
     1981 gtcttttcct tacccatggt tgtttatgtt cggatgtgat gtgagaactg tatcctagca
     2041 agattttaaa aggaagtata tgaaagaaga acctcagtgg caaatcctaa ccttttatat
     2101 ttctctacag gggcgcggcg tggggacaat tcaacgcgtc tgtgagggga gcgtttccct
     2161 gctcgcaggt ctgcagcgag gagccgtaat ttttgcttcg cgccgtgcgg ccatcaaaat
     2221 gtatggatgc aaatgattat acatggggat gtatgggcta aatgtacggg cgacagtcac
     2281 atcatgcccc tgagctgcgc acgtcaagac tgtcaaggag ggtattctgg gcctccatgt
     2341 cgctggccgg gtgacccggc ggggacgagg caagctaaac agatctcgcc ataaaagaca
     2401 gaataagtca tcagcggttg tttcatttcc tatatttttt ttttattttt ttatttttta
     2461 ataagggaaa atttaacgtc taaggataca gaagattgtt agcacattaa agtaataaag
     2521 gcttaagtag taagtgcctt agcatgttat tgtatttcaa aggacataat ctaaaataat
     2581 aacaatatca tttctcacaa gttattcaat tttctttttt tttctaataa tatcaagaat
     2641 gtattatttg tttgacataa gtcaactaat ttatttaata tgctggatta atcttgcaga
     2701 catgtaaatt aacaagtttt agtcaaataa cgttgaagtt tcaatgaact caaataattt
     2761 ctcttttttt ttatataacc atatgtctaa tctgatttat attttccgca gggatcaact
     2821 gaagttatga catttggatt ggatcactta taaccttggt cgccaaataa tacaaaaatc
     2881 agcgttataa aacaaagaag gtttttgtta agaaattaat cctctttctt gataagaaag
     2941 ttgaaccgaa attgcagata ctgatatatg aaaataatac ccacaatttt gggaatagcg
     3001 caagcctcaa tttaaacaat aggtgaggac acatgataat gacctcaatg attgttagaa
     3061 gaaaagagcc tcattacaaa atcgaaaaat gaatggttgg gtacaagttt ccaaaacatg
     3121 gtaaagtgga ctttgcgtat gagacgtaaa tagaaaaaaa cacttgttat atgttttcta
     3181 gaattattgt tgtctcttta tggttggatg atgcaaaata gtaatttcgg ttagttgctg
     3241 taaaacacca cgagacaaat agatatggat atttattaaa tcaggaaaaa cgtaactctc
     3301 ggctactgga tggttcagtc acccaacgat tactggggag agaaaacagg gcaaaagcaa
     3361 agcttaaagg aatccgattg tcattcggca atgtgcagcg aaactaaaaa ccggataatg
     3421 gacctgttaa tcgaaacatt gaagatatat aaaggaagag gaatcctggc atatcatcaa
     3481 ttgaataagt tgaattaatt atttcaatct cattctcact ttctgactta tagtcgcttt
     3541 gttaaatcat gtctttaatt aacggtagtg gagttagcaa gggcgaggag accatcatga
     3601 gcgtgatcaa gccggacatg aagatcaagc tgcgcatgga gggcaacgtg aacggccatg
     3661 cctttgtgat cgagggcgag ggcagcggta agccgtttga gggcatccag accatcgacc
     3721 tggaggttaa ggaaggcgca ccgctgccgt ttgcctacga catcctgacc accgcattcc
     3781 actacggcaa ccgcgtgttc accaagtacc cgcgcaagat cccggactac ttcaagcaga
     3841 gcttcccgga gggctacagt tgggaacgca gcatgaccta cgaggacggc ggtatctgca
     3901 acgccaccaa cgacatcacc atggaagaag atagcttcat caacaagatc cacttcaagg
     3961 gcacaaactt cccgccgaat ggtccggtta tgcagaagcg caccgttggc tgggaggtga
     4021 gcaccgagaa gatgtatgtg cgcgacggtg tgctgaaggg cgacgtgaag atgaagctgc
     4081 tgctgaaggg tggcagccac taccgctgcg acttccgcac cacctacaaa gttaagcaaa
     4141 aggcagtgaa gttaccgaag gcccacttcg tggaccaccg catcgaaatc ctgagccacg
     4201 acaaggacta taacaaagtg aagctgtacg agcacgccgt ggcccgtaac agcaccgaca
     4261 gcatggatga gctgtacaaa taaggcgcgc cacttctaaa taagcgaatt tcttatgatt
     4321 tatgattttt attattaaat aagttataaa aaaaataagt gtatacaaat tttaaagtga
     4381 ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac tctttcctgt aggtcaggtt
     4441 gctttctcag gtatagtatg aggtcgctct tattgaccac acctctaccg gcagatccgc
     4501 tagggataac agggtaatat agatccgtcg acctgcagcg tacgaagctt cagctggcgg
     4561 ccgcgttcta tagtgtcacc taaatcgtat gtgtatgata cataaggtta tgtattaatt
     4621 gtagccgcgt tctaacgaca atatgtccat atggtgcact ctcagtacaa tctgctctga
     4681 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     4741 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     4801 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     4861 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     4921 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     4981 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     5041 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5101 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5161 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5221 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     5281 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5341 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5401 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     5461 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     5521 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     5581 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     5641 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     5701 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     5761 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     5821 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     5881 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     5941 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6001 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa a
//
