LOCUS       sequence-82826-e        5103 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gene deletion with URA3-kanMX6 double marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5103)
  AUTHORS   Onishi M, Ko N, Nishihama R, Pringle JR
  TITLE     Distinct roles of Rho1, Cdc42, and Cyk3 in septum formation and 
            abscission during yeast cytokinesis.
  JOURNAL   J Cell Biol. 2013 Jul 22;202(2):311-29. doi: 10.1083/jcb.201302001.
  PUBMED    23878277
REFERENCE   2  (bases 1 to 5103)
  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"; 
            journalName: "J Cell Biol"; date: "2013-07-22- 22"; volume: "202"; 
            issue: "2"; pages: "311-29"
COMMENT     Sequence Label: pFA6a-URA3-KanMX6
FEATURES             Location/Qualifiers
     source          1..5103
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        117..332
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             333..1136
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     gene            1280..2636
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        1280..1623
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1624..2433
                     /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(1691..1710)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2439..2636
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2740..2759)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2741..2759)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2846..2863)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3017..3605)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3097..3116)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3776..4567,4568..4636))
                     /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     4399..4418
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4637..4741)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4809..4827
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4865..4887)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        join(5087..5103,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(5087..5103,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg acggatcccc gggttaatta
       61 aggcgcgcct atgcggcatc agagcagatt gtactgagag tgcaccatac caccttttca
      121 attcatcatt ttttttttat tctttttttt gatttcggtt tctttgaaat ttttttgatt
      181 cggtaatctc cgaacagaag gaagaacgaa ggaaggagca cagacttaga ttggtatata
      241 tacgcatatg tagtgttgaa gaaacatgaa attgcccagt attcttaacc caactgcaca
      301 gaacaaaaac ctgcaggaaa cgaagataaa tcatgtcgaa agctacatat aaggaacgtg
      361 ctgctactca tcctagtcct gttgctgcca agctatttaa tatcatgcac gaaaagcaaa
      421 caaacttgtg tgcttcattg gatgttcgta ccaccaagga attactggag ttagttgaag
      481 cattaggtcc caaaatttgt ttactaaaaa cacatgtgga tatcttgact gatttttcca
      541 tggagggcac agttaagccg ctaaaggcat tatccgccaa gtacaatttt ttactcttcg
      601 aagacagaaa atttgctgac attggtaata cagtcaaatt gcagtactct gcgggtgtat
      661 acagaatagc agaatgggca gacattacga atgcacacgg tgtggtgggc ccaggtattg
      721 ttagcggttt gaagcaggcg gcagaagaag taacaaagga acctagaggc cttttgatgt
      781 tagcagaatt gtcatgcaag ggctccctat ctactggaga atatactaag ggtactgttg
      841 acattgcgaa gagcgacaaa gattttgtta tcggctttat tgctcaaaga gacatgggtg
      901 gaagagatga aggttacgat tggttgatta tgacacccgg tgtgggttta gatgacaagg
      961 gagacgcatt gggtcaacag tatagaaccg tggatgatgt ggtctctaca ggatctgaca
     1021 ttattattgt tggaagagga ctatttgcaa agggaaggga tgctaaggta gagggtgaac
     1081 gttacagaaa agcaggctgg gaagcatatt tgagaagatg cggccagcaa aactaaaaaa
     1141 ctgtattata agtaaatgca tgtatactaa actcacaaat tagagcttca atttaattat
     1201 atcagttatt accctgcggt gtgaaatacc gcacagatct gtttagcttg cctcgtcccc
     1261 gccgggtcac ccggccagcg acatggaggc ccagaatacc ctccttgaca gtcttgacgt
     1321 gcgcagctca ggggcatgat gtgactgtcg cccgtacatt tagcccatac atccccatgt
     1381 ataatcattt gcatccatac attttgatgg ccgcacggcg cgaagcaaaa attacggctc
     1441 ctcgctgcag acctgcgagc agggaaacgc tcccctcaca gacgcgttga attgtcccca
     1501 cgccgcgccc ctgtagagaa atataaaagg ttaggatttg ccactgaggt tcttctttca
     1561 tatacttcct tttaaaatct tgctaggata cagttctcac atcacatccg aacataaaca
     1621 accatgggta aggaaaagac tcacgtttcg aggccgcgat taaattccaa catggatgct
     1681 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat
     1741 cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt
     1801 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt
     1861 ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc
     1921 cccggcaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt
     1981 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt
     2041 aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt
     2101 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa
     2161 atgcataagc ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt
     2221 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga
     2281 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct
     2341 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg
     2401 cagtttcatt tgatgctcga tgagtttttc taatcagtac tgacaataaa aagattcttg
     2461 ttttcaagaa cttgtcattt gtatagtttt tttatattgt agttgttcta ttttaatcaa
     2521 atgttagcgt gatttatatt ttttttcgcc tcgacatcat ctgcccagat gcgaagttaa
     2581 gtgcgcagaa agtaatatca tgcgtcaatc gtatgtgaat gctggtcgct atactgctgt
     2641 cgattcgata ctaacgccgc catccagttt aaacgagctc gaattcatcg atgatatcag
     2701 atccactagt ggcctatgcg gccgcggatc tgccggtctc cctatagtga gtcgtattaa
     2761 tttcgataag ccaggttaac ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     2821 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     2881 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     2941 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     3001 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     3061 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     3121 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     3181 ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg
     3241 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     3301 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     3361 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     3421 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     3481 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     3541 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     3601 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     3661 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     3721 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     3781 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     3841 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     3901 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     3961 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     4021 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     4081 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     4141 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     4201 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     4261 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     4321 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     4381 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     4441 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     4501 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     4561 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     4621 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     4681 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     4741 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     4801 cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg
     4861 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     4921 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
     4981 actatgcggc atcagagcag attgtactga gagtgcacca tatggacata ttgtcgttag
     5041 aacgcggcta caattaatac ataaccttat gtatcataca catacgattt aggtgacact
     5101 ata
//
