LOCUS       sequence-203253-        6033 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 6033)
  AUTHORS   Davidson R, Pontasch JA, Wu JQ
  TITLE     Sbg1 Is a Novel Regulator for the Localization of the beta-Glucan 
            Synthase Bgs1 in Fission Yeast.
  JOURNAL   PLoS One. 2016 Nov 29;11(11):e0167043. doi: 
            10.1371/journal.pone.0167043. eCollection 2016.
  PUBMED    27898700
REFERENCE   2  (bases 1 to 6033)
  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: "PLoS One.";
            date: "2016-11-29"; volume: "11(11):e0167043. doi"; pages: " 
            10.1371/journal.pone.0167043. eCollection 2016"
COMMENT     Sequence Label: pFA6a-kanMX6-P81nmt1-mECitrine
FEATURES             Location/Qualifiers
     source          1..6033
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        4..22
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4..21
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     terminator      complement(98..285)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     206..230
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(349..369)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     964..992
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     promoter        complement(1034..2185)
                     /label=nmt1 P81 promoter
                     /note="mutant nmt1 promoter from Schizosaccharomyces pombe,
                     conferring weak thiamine-repressible expression"
     gene            2230..3586
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        2230..2573
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2574..3383
                     /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(2641..2660)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      3389..3586
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3690..3709)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3691..3709)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3796..3813)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3967..4555)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4047..4066)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4726..5517,5518..5586))
                     /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     5349..5368
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5587..5691)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5759..5777
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5815..5837)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5937..5956
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 acgatttagg tgacactata gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg
       61 acggatctat attaccctgt tatccctagc ggatctgccg gtagaggtgt ggtcaataag
      121 agcgacctca tactatacct gagaaagcaa cctgacctac aggaaagagt tactcaagaa
      181 taagaatttt cgttttaaaa cctaagagtc actttaaaat ttgtatacac ttattttttt
      241 tataacttat ttaataataa aaatcataaa tcataagaaa ttcgcttatt tagaagtggc
      301 gcgccctatt tgtatagttc atccatgcca tgtgtaatcc cagcagctgt tacaaactca
      361 agaaggacca tgtggtctct cttttcgttg ggatctttcg aaagcttaga ttgataggac
      421 aggtaatggt tgtctggtaa aaggacaggg ccatcgccaa ttggagtatt ttgttgataa
      481 tggtctgcta gttgaacgct tccatcttca atgttgtgtc taattttgaa gttaactttg
      541 attccattct tttgtttgtc tgccatgatg tatacattgt gtgagttata gttgtattcc
      601 aatttgtgtc caagaatgtt tccatcttct ttaaaatcaa taccttttaa ctcgattcta
      661 ttaacaaggg tatcaccttc aaacttgact tcagcacgtg tcttgtagtt cccgtcatct
      721 ttgaaaaata tagttctttc ctgtacataa ccttcgggca tggcactctt gaaaaagtca
      781 tgccgtttca tatgatctgg gtatctagca aagcacatta gaccataacc caaagtagtg
      841 acaagtgttg gccatggaac aggtagtttt ccagtagtgc aaataaattt aagggtaagt
      901 tttccgtatg ttgcatcacc ttcaccctct ccactgacag aaaatttgtg cccattaaca
      961 tcaccatcta attcaacaag aattgggaca actccagtga aaagttcttc tcctttactg
     1021 ttaattaaag acatgattta acaaagcgac tataagtcag aaagtgagaa tgagattgaa
     1081 ataattaatt caacttattc aattgatgat atgccaggat tcctcttcca tcttcaatgt
     1141 ttcgattaac aggtccatta tccggttttt agtttcgctg cacattgccg aatgacaatc
     1201 ggattccttt aagctttgct tttgccctgt tttctctccc cagtaatcgt tgggtgactg
     1261 aaccatccag tagccgagag ttacgttttt cctgatttaa taaatatcca tatctatttg
     1321 tctcgtggtg ttttacagca actaaccgaa attactattt tgcatcatcc aaccataaag
     1381 agacaacaat aattctagaa aacatataac aagtgttttt ttctatttac gtctcatacg
     1441 caaagtccac tttaccatgt tttggaaact tgtacccaac cattcatttt tcgattttgt
     1501 aatgaggctc ttttcttcta acaatcattg aggtcattat catgtgtcct cacctattgt
     1561 ttaaattgag gcttgcgcta ttcccaaaat tgtgggtatt attttcatat atcagtatct
     1621 gcaatttcgg ttcaactttc ttatcaagaa agaggattaa tttcttaaca aaaaccttct
     1681 ttgttttata acgctgattt ttgtattatt tggcgaccaa ggttataagt gatccaatcc
     1741 aaatgtcata acttcagttg atccctgcgg aaaatataaa tcagattatg gttatataaa
     1801 aaaaaagaga aattatttga gttcattgaa acttcaacgt tatttgacta aaacttgtta
     1861 atttacatgt ctgcaagatt aatccagcat attaaataaa ttagttgact tatgtcaaac
     1921 aaataataca ttcttgatat tattagaaaa aaaaaagaaa attgaataac ttgtgagaaa
     1981 tgatattgtt attattttag attatgtcct ttgaaataca ataacatgct aaggcactta
     2041 ctacttaagc ctttattact ttaatgtgct aacaatcttc tgtatcctta gacgttaaat
     2101 tttcccttat taaaaaataa aaaaataaaa aaaaaatata ggaaatgaaa caaccgctga
     2161 tgacttattc tgtcttttat ggcgagatct gtttagcttg cctcgtcccc gccgggtcac
     2221 ccggccagcg acatggaggc ccagaatacc ctccttgaca gtcttgacgt gcgcagctca
     2281 ggggcatgat gtgactgtcg cccgtacatt tagcccatac atccccatgt ataatcattt
     2341 gcatccatac attttgatgg ccgcacggcg cgaagcaaaa attacggctc ctcgctgcag
     2401 acctgcgagc agggaaacgc tcccctcaca gacgcgttga attgtcccca cgccgcgccc
     2461 ctgtagagaa atataaaagg ttaggatttg ccactgaggt tcttctttca tatacttcct
     2521 tttaaaatct tgctaggata cagttctcac atcacatccg aacataaaca accatgggta
     2581 aggaaaagac tcacgtttcg aggccgcgat taaattccaa catggatgct gatttatatg
     2641 ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg
     2701 ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg
     2761 ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca
     2821 agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccggcaaaa
     2881 cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg
     2941 cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc
     3001 gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg
     3061 attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataagc
     3121 ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta
     3181 tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc
     3241 gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga
     3301 aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt
     3361 tgatgctcga tgagtttttc taatcagtac tgacaataaa aagattcttg ttttcaagaa
     3421 cttgtcattt gtatagtttt tttatattgt agttgttcta ttttaatcaa atgttagcgt
     3481 gatttatatt ttttttcgcc tcgacatcat ctgcccagat gcgaagttaa gtgcgcagaa
     3541 agtaatatca tgcgtcaatc gtatgtgaat gctggtcgct atactgctgt cgattcgata
     3601 ctaacgccgc catccagttt aaacgagctc gaattcatcg atgatatcag atccactagt
     3661 ggcctatgcg gccgcggatc tgccggtctc cctatagtga gtcgtattaa tttcgataag
     3721 ccaggttaac ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3781 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     3841 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     3901 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     3961 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4021 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4081 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4141 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4201 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4261 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4321 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4381 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     4441 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     4501 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     4561 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     4621 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     4681 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     4741 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4801 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4861 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     4921 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     4981 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5041 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5101 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5161 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5221 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5281 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5341 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     5401 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     5461 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     5521 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     5581 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     5641 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     5701 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     5761 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
     5821 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     5881 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
     5941 atcagagcag attgtactga gagtgcacca tatggacata ttgtcgttag aacgcggcta
     6001 caattaatac ataaccttat gtatcataca cat
//
