LOCUS       sequence-417278-        7957 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  episomal bioluminescent reporter plasmid for S. stipitis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7957)
  AUTHORS   Reichard WD, Smith SE, Robertson JB
  TITLE     BLINCAR: a reusable bioluminescent and Cas9-based genetic toolset 
            for repeatedly modifying wild-type Scheffersomyces stipitis.
  JOURNAL   mSphere. 2023 Aug 24;8(4):e0022423. doi: 10.1128/msphere.00224-23. 
            Epub 2023 Jun 22.
  PUBMED    37345937
REFERENCE   2  (bases 1 to 7957)
  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: "mSphere."; 
            date: "2023-08-24"; volume: "8(4):e0022423. doi"; pages: " 
            10.1128/msphere.00224-23. Epub 2023 Jun 22"
COMMENT     Sequence Label: pWR23
FEATURES             Location/Qualifiers
     source          1..7957
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             777..1586
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             2834..4462
                     /codon_start=1
                     /product="green-emitting variant of click beetle 
                     luciferase"
                     /label=CBG99luc
                     /note="human codon-optimized"
                     /translation="MVKREKNVIYGPEPLHPLEDLTAGEMLFRALRKHSHLPQALVDVV
                     GDESLSYKEFFEATVLLAQSLHNCGYKMNDVVSICAENNTRFFIPVIAAWYIGMIVAPV
                     NESYIPDELCKVMGISKPQIVFTTKNILNKVLEVQSRTNFIKRIIILDTVENIHGCESL
                     PNFISRYSDGNIANFKPLHFDPVEQVAAILCSSGTTGLPKGVMQTHQNICVRLIHALDP
                     RVGTQLIPGVTVLVYLPFFHAFGFSITLGYFMVGLRVIMFRRFDQEAFLKAIQDYEVRS
                     VINVPSVILFLSKSPLVDKYDLSSLRELCCGAAPLAKEVAEVAAKRLNLPGIRCGFGLT
                     ESTSANIHSLGDEFKSGSLGRVTPLMAAKIADRETGKALGPNQVGELCIKGPMVSKGYV
                     NNVEATKEAIDDDGWLHSGDFGYYDEDEHFYVVDRYKELIKYKGSQVAPAELEEILLKN
                     PCIRDVAVVGIPDLEAGELPSAFVVKQPGKEITAKEVYDYLAERVSHTKYLRGGVRFVD
                     SIPRNVTGKITRKELLKQLLEKAGG"
     RBS             3784..3792
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     rep_origin      complement(6202..6790)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6282..6301)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6961..7752,7753..7821))
                     /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     7584..7603
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7822..7926)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccaatgatac cggtgcgatc gtgggcccta gtacttccct cgagccgtac acttattgtt
       61 aactatgaaa catatactta tggacaaggt tcactgattt ggactgaaaa gcaaaaacga
      121 gaaatgagtc aacgtctgag atcaatccct agaccacccg ggaaggtcat gtgtttcatg
      181 aagtacgata aggttggtaa ccgattgact cattggttcg tggcggagaa gtacgcagag
      241 taaaaccggg gccgattcgt ggtaaattct ggaatgatcc agaggcgcga catttatgca
      301 gacaatttgt gttttgtcgc aaacgatgtt atagcgaaat ttttcactct gtcagataaa
      361 tggattttgt caaaaggggg aagtagaagg agaatgggcc cgagatgttc tgccaaattc
      421 tcagtagcat aatgtgaaag aagcccttac attgtccagc ctctggcatc attaaaaacc
      481 gtagcggaaa ccaattgtct ctgttcttcc ctggcacacc ctggtagccc cacccagttg
      541 tagtacatct cacacgctgg caacttggga caatcagcaa cttttttttc ttttaatttt
      601 ttcagcgcga cattttgcct cttctgcgag aacagacttt ttcacctcca tctcaccccc
      661 ctttgcactt atataaattg gaccagttcc tcccattgta gaaaaaattt tgctggacct
      721 ttttctcttt tttttgtcct ttagtttcat acaatctaag tctatctaca tgtacaatgg
      781 gtaaggaaaa gactcacgtt tcgaggccga gattaaattc caacatggat gctgatttat
      841 atgggtataa atgggctaga gataatgtcg ggcaatcagg tgcgacaatc tatagattgt
      901 atgggaagcc cgatgcgcca gagttgtttt tgaaacatgg caaaggtagc gttgccaatg
      961 atgttacaga tgagatggtc agactaaact ggttgacgga atttatgcct cttccgacca
     1021 tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg atccccggca
     1081 aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgt
     1141 tggcagtgtt cttgagaaga ttgcattcga ttcctgtttg taattgtcct tttaacagcg
     1201 atagagtatt tcgtctcgct caggcgcaat caagaatgaa taacggtttg gttgatgcga
     1261 gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata
     1321 agttgttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc
     1381 ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggaagagtc ggaatcgcag
     1441 acagatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac
     1501 agaaaagact ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc
     1561 atttgatgct cgatgagttt ttctaagcgg ccgctaacca cttgcaaaat cctttgtatt
     1621 cttgttctgc aaacattttg tccaatctct tatctttcta cgatgttgaa gaacatacat
     1681 ttttgttagt ccagcttgtt atcctttttt tattaaataa aaaattttat tgttaaagtt
     1741 gtttttcttc tttgtctgtc ctgttttaac tcatatatat tctatatatt acagaaagaa
     1801 agtagatcat ctaaaataaa catttatttg atgagacaaa gtaggttttt tagaatacaa
     1861 tgtagcatcc tctaatatac aatagagttg ttttataaaa gcgatacatt taatattatg
     1921 acaggactgg tgaagtgtat agaattgtgc taatctggca aacgaaccca agaagacgtt
     1981 aaagcaaata gtgcattttg tattacaatc aagttcgtcg accatgcatt agatcttgca
     2041 tgcctctaga acctaggtgg cgccacagct gtggatcctc gttagtattt ccgtgaaggc
     2101 agccattgtg aaaattgaac atcacctgag gtattttagc caccagaagc ggcggtacgg
     2161 aagaaagtgt gtacgatggt tggtggtgga attgcgtgca tgcctgatgg ggcaatatta
     2221 attagataga gctttggtga tattagtgga taatagaatt cacagagaag acatcaggag
     2281 caatttccaa gagccattga tgatgtaatt gccccaacag caagattcag atctgacaat
     2341 tgaccaccgt tttgtagaag caaaaaatcg tagattatca ccaagagggt ttttcaccga
     2401 accagcaaat agaaactatg ccgtagaact cgcccaggct tttttgctag cactttccag
     2461 cagtagaacc gtccaattaa gtcaacagga accattgagg tcgagcccaa ccacctgaac
     2521 cccctcacgg tcgtgtccct attattgatc cagagggtgc cagtttcggt agccaatatt
     2581 ggttcatggg tttctatggc ccggagtgag tttgcaggtt ggccccggcg ccgtctgcag
     2641 gatgggagtt atagcggcca aacttcacat ttcgaaatcc tgctgcagcc aatctgaaga
     2701 attaatataa attcgtgtcg aatcgccgtc tgtgaaattt cagtacttga ttttcttttc
     2761 ttcttctttt tctcttttgt ttcttcagaa tcaatacaca ttttttcttc cctataaaca
     2821 attcatcggt accatggtga agcgtgagaa aaatgtcatc tatggccctg agcctctcca
     2881 tcctttggag gatttgactg ccggcgaaat gttgtttcgt gctctccgca agcactctca
     2941 tttgcctcaa gccttggtcg atgtggtcgg cgatgaatct ttgagctaca aggagttttt
     3001 tgaggcaacc gtcttgttgg ctcagtccct ccacaattgt ggctacaaga tgaacgacgt
     3061 cgttagtatc tgtgctgaaa acaatacccg tttcttcatt ccagtcatcg ccgcatggta
     3121 tatcggtatg atcgtggctc cagtcaacga gagctacatt cccgacgaat tgtgtaaagt
     3181 catgggtatc tctaagccac agattgtctt caccactaag aatattttga acaaagtctt
     3241 ggaagtccaa agccgcacca actttattaa gcgtatcatc atcttggaca ctgtggagaa
     3301 tattcacggt tgcgaatctt tgcctaattt catctctcgc tattcagacg gcaacatcgc
     3361 aaactttaaa ccactccact tcgaccctgt ggaacaagtt gcagccattt tgtgtagcag
     3421 cggtactact ggactcccaa agggagtcat gcagacccat caaaacattt gcgtgcgttt
     3481 gatccatgct ctcgatccac gtgtgggcac tcagttgatt cctggtgtca ccgtcttggt
     3541 ctacttgcct ttcttccatg ctttcggctt tagcattact ttgggttact ttatggtcgg
     3601 tctccgcgtg attatgttcc gccgttttga tcaggaggct ttcttgaaag ccatccaaga
     3661 ttatgaagtc cgcagtgtca tcaacgtgcc tagcgtgatc ttgtttttgt ctaagagccc
     3721 actcgtggac aagtacgact tgtcttcatt gcgtgaattg tgttgcggtg ccgctccatt
     3781 ggctaaggag gtcgctgaag tggccgccaa acgcttgaat cttccaggta ttcgttgtgg
     3841 cttcggcctc accgaatcta ccagcgctaa cattcactct ctcggtgatg agtttaagag
     3901 cggctctttg ggccgtgtca ctccactcat ggctgctaag atcgctgatc gcgaaactgg
     3961 taaggctttg ggtccgaacc aagtgggcga gttgtgtatc aaaggcccta tggtgagcaa
     4021 gggttatgtc aataacgttg aagctaccaa ggaggccatc gacgacgacg gctggttgca
     4081 ttctggtgat tttggatatt acgacgaaga tgagcatttt tacgtcgtgg atcgttacaa
     4141 ggagttgatc aaatacaagg gtagccaggt tgctccagct gagttggagg agattttgtt
     4201 gaaaaatcca tgcattcgcg atgtcgctgt ggtcggcatt cctgatttgg aggccggcga
     4261 attgccttct gctttcgttg tcaagcagcc tggtaaagaa attaccgcca aagaagtgta
     4321 tgattacttg gctgaacgtg tgagccatac taagtacttg cgtggcggcg tgcgttttgt
     4381 tgactccatc cctcgtaacg taacaggcaa aattacccgc aaggagttgt tgaaacaatt
     4441 gttggagaag gccggcggtt agcttaagca ttttagacaa gtgcctattt attcagattg
     4501 attttttata ttgaatatgg atgttgatta cgacttgtgt tacgaagcat gaacatgtga
     4561 tttgaaaaag tgtttacgaa taaaagaact ttttttgcaa ccgttgctag ttgaagtgta
     4621 gactctgaat gcgaagggtt gcaaatgtgc ccagtagaaa aaatattcca tatttttctg
     4681 aacttaattc aatgaaatac gagtattact acaactgcca atatcggaac aatttaacga
     4741 gagcaagcta tataaaatga atgttcattt acacaagcta gaaccaaaac ttgaattaaa
     4801 atttaacata actaaatgta actagataaa agagagacat aaccaaacgt tcagaaggca
     4861 gaagaattca gttaactcct gcaggaatcg attggccggc ctcacgtgga cgcgttatta
     4921 atcgtttaaa cctacgtaac cgcggcttaa ttaaagctag ccagcgcttg agctcagtat
     4981 aggatatggt gatttagcaa aatatgcctc tgtttttttg gtaagttgta gtaattcgga
     5041 atttttcgca acttttccag aaatcgcttt atgccaaaca tattatacgc aaattattta
     5101 taatcatgta atatgttaag tctctggaat ttcatgtcag cagtgaaacg aagttgcaca
     5161 gtctatagca cccttaacgc tgaagtattg actaaatgtg tctatttttc tcttcgtaca
     5221 ttttagtcag attgtcgaaa tctaaatctc tacaggctgc aaccaaagac atcttgctga
     5281 ttcgtatctc atttctaact cagcaaaatt ttattatcca gcagacaaca attatctcct
     5341 ttcttacatg atacttagac ttctaatctc ctagtggata agaaaaataa atccaaagaa
     5401 tggaaaatgt aaagctgcgc tggtgatgtc cgctaaggtc ataaactcaa gctacattgc
     5461 gtcgaatatt ttgctacgat tgctcagaaa taaaatccat tgtatatcat gagggtcaat
     5521 aataataata atcaacgagt ccgattgctg aaccacccaa taaatatcaa tcatggtctt
     5581 acaagcaaac acaacataca ataattatcg gggcggtcca acaatgtcgg ctcgacttgt
     5641 actgtatgca cctagcgttt ttcatctggc atttatccga gtacccctaa acgctaaagc
     5701 gtagagaata atgtaacctt atcttgccgg agcaactaac aatcgccttg aacacaaact
     5761 gggttgctcc cctgttctcg aagtcgaaga gtagctcctt ctagctccag tgaaaatgta
     5821 cagatacgca gagatggtga ctgtacctaa acagactccg tttgcaccta ctccgtcaga
     5881 acaatagctc tacaataaag cctcccacca cgctccactt acatatgcat atgcacatac
     5941 attgtaagag aagtatgttg gccatgtaga cacaaacaag atgtcggcga cctacagatt
     6001 cttttatcca gccaatggag agagaaggta cttgtcagag aagaatgcat gcaccgggaa
     6061 ataatgcggt gtctggggct tttggatgag ttaaaatact tctgaccttg tagacaccgc
     6121 agagagctcc atcggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     6181 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     6241 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     6301 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     6361 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     6421 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     6481 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     6541 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     6601 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     6661 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     6721 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     6781 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     6841 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     6901 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     6961 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     7021 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     7081 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     7141 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     7201 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     7261 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     7321 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     7381 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     7441 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     7501 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     7561 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     7621 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     7681 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     7741 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     7801 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     7861 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     7921 tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
