LOCUS       sequence-409735-        8176 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 8176)
  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 8176)
  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: pWR9
FEATURES             Location/Qualifiers
     source          1..8176
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             777..1805
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             3053..4681
                     /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             4003..4011
                     /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(6421..7009)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6501..6520)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7180..7971,7972..8040))
                     /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     7803..7822
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8041..8145)
                     /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 gtaaaaagcc tgaactcacc gcgacctctg tcgagaagtt tttgatcgaa aagttcgaca
      841 gcgtctccga cttgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg
      901 taggagggcg tggatatgtc ttgcgggtaa atagctgcgc cgatggtttc tacaaagatc
      961 gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg
     1021 gggagttcag cgagagcttg acctattgca tctcccgccg tgcacagggt gtcacgttgc
     1081 aagacttgcc tgaaaccgaa ttgcccgctg ttttgcagcc ggtcgcggag gccatggatg
     1141 cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa
     1201 tcggtcaata cactacatgg cgtgatttca tttgcgcgat tgctgatccc catgtgtatc
     1261 actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagt
     1321 tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct
     1381 ccaacaatgt cttgacggac aatggccgca taacagcggt cattgactgg agcgaggcga
     1441 tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt
     1501 gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccac
     1561 ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg
     1621 gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag
     1681 ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct
     1741 gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg
     1801 aataagcggc cgctaaccac ttgcaaaatc ctttgtattc ttgttctgca aacattttgt
     1861 ccaatctctt atctttctac gatgttgaag aacatacatt tttgttagtc cagcttgtta
     1921 tccttttttt attaaataaa aaattttatt gttaaagttg tttttcttct ttgtctgtcc
     1981 tgttttaact catatatatt ctatatatta cagaaagaaa gtagatcatc taaaataaac
     2041 atttatttga tgagacaaag taggtttttt agaatacaat gtagcatcct ctaatataca
     2101 atagagttgt tttataaaag cgatacattt aatattatga caggactggt gaagtgtata
     2161 gaattgtgct aatctggcaa acgaacccaa gaagacgtta aagcaaatag tgcattttgt
     2221 attacaatca agttcgtcga ccatgcatta gatcttgcat gcctctagaa cctaggtggc
     2281 gccacagctg tggatcctcg ttagtatttc cgtgaaggca gccattgtga aaattgaaca
     2341 tcacctgagg tattttagcc accagaagcg gcggtacgga agaaagtgtg tacgatggtt
     2401 ggtggtggaa ttgcgtgcat gcctgatggg gcaatattaa ttagatagag ctttggtgat
     2461 attagtggat aatagaattc acagagaaga catcaggagc aatttccaag agccattgat
     2521 gatgtaattg ccccaacagc aagattcaga tctgacaatt gaccaccgtt ttgtagaagc
     2581 aaaaaatcgt agattatcac caagagggtt tttcaccgaa ccagcaaata gaaactatgc
     2641 cgtagaactc gcccaggctt ttttgctagc actttccagc agtagaaccg tccaattaag
     2701 tcaacaggaa ccattgaggt cgagcccaac cacctgaacc ccctcacggt cgtgtcccta
     2761 ttattgatcc agagggtgcc agtttcggta gccaatattg gttcatgggt ttctatggcc
     2821 cggagtgagt ttgcaggttg gccccggcgc cgtctgcagg atgggagtta tagcggccaa
     2881 acttcacatt tcgaaatcct gctgcagcca atctgaagaa ttaatataaa ttcgtgtcga
     2941 atcgccgtct gtgaaatttc agtacttgat tttcttttct tcttcttttt ctcttttgtt
     3001 tcttcagaat caatacacat tttttcttcc ctataaacaa ttcatcggta ccatggtgaa
     3061 gcgtgagaaa aatgtcatct atggccctga gcctctccat cctttggagg atttgactgc
     3121 cggcgaaatg ttgtttcgtg ctctccgcaa gcactctcat ttgcctcaag ccttggtcga
     3181 tgtggtcggc gatgaatctt tgagctacaa ggagtttttt gaggcaaccg tcttgttggc
     3241 tcagtccctc cacaattgtg gctacaagat gaacgacgtc gttagtatct gtgctgaaaa
     3301 caatacccgt ttcttcattc cagtcatcgc cgcatggtat atcggtatga tcgtggctcc
     3361 agtcaacgag agctacattc ccgacgaatt gtgtaaagtc atgggtatct ctaagccaca
     3421 gattgtcttc accactaaga atattttgaa caaagtcttg gaagtccaaa gccgcaccaa
     3481 ctttattaag cgtatcatca tcttggacac tgtggagaat attcacggtt gcgaatcttt
     3541 gcctaatttc atctctcgct attcagacgg caacatcgca aactttaaac cactccactt
     3601 cgaccctgtg gaacaagttg cagccatttt gtgtagcagc ggtactactg gactcccaaa
     3661 gggagtcatg cagacccatc aaaacatttg cgtgcgtttg atccatgctc tcgatccacg
     3721 tgtgggcact cagttgattc ctggtgtcac cgtcttggtc tacttgcctt tcttccatgc
     3781 tttcggcttt agcattactt tgggttactt tatggtcggt ctccgcgtga ttatgttccg
     3841 ccgttttgat caggaggctt tcttgaaagc catccaagat tatgaagtcc gcagtgtcat
     3901 caacgtgcct agcgtgatct tgtttttgtc taagagccca ctcgtggaca agtacgactt
     3961 gtcttcattg cgtgaattgt gttgcggtgc cgctccattg gctaaggagg tcgctgaagt
     4021 ggccgccaaa cgcttgaatc ttccaggtat tcgttgtggc ttcggcctca ccgaatctac
     4081 cagcgctaac attcactctc tcggtgatga gtttaagagc ggctctttgg gccgtgtcac
     4141 tccactcatg gctgctaaga tcgctgatcg cgaaactggt aaggctttgg gtccgaacca
     4201 agtgggcgag ttgtgtatca aaggccctat ggtgagcaag ggttatgtca ataacgttga
     4261 agctaccaag gaggccatcg acgacgacgg ctggttgcat tctggtgatt ttggatatta
     4321 cgacgaagat gagcattttt acgtcgtgga tcgttacaag gagttgatca aatacaaggg
     4381 tagccaggtt gctccagctg agttggagga gattttgttg aaaaatccat gcattcgcga
     4441 tgtcgctgtg gtcggcattc ctgatttgga ggccggcgaa ttgccttctg ctttcgttgt
     4501 caagcagcct ggtaaagaaa ttaccgccaa agaagtgtat gattacttgg ctgaacgtgt
     4561 gagccatact aagtacttgc gtggcggcgt gcgttttgtt gactccatcc ctcgtaacgt
     4621 aacaggcaaa attacccgca aggagttgtt gaaacaattg ttggagaagg ccggcggtta
     4681 gcttaagcat tttagacaag tgcctattta ttcagattga ttttttatat tgaatatgga
     4741 tgttgattac gacttgtgtt acgaagcatg aacatgtgat ttgaaaaagt gtttacgaat
     4801 aaaagaactt tttttgcaac cgttgctagt tgaagtgtag actctgaatg cgaagggttg
     4861 caaatgtgcc cagtagaaaa aatattccat atttttctga acttaattca atgaaatacg
     4921 agtattacta caactgccaa tatcggaaca atttaacgag agcaagctat ataaaatgaa
     4981 tgttcattta cacaagctag aaccaaaact tgaattaaaa tttaacataa ctaaatgtaa
     5041 ctagataaaa gagagacata accaaacgtt cagaaggcag aagaattcag ttaactcctg
     5101 caggaatcga ttggccggcc tcacgtggac gcgttattaa tcgtttaaac ctacgtaacc
     5161 gcggcttaat taaagctagc cagcgcttga gctcwstryr gkrtctacaa ggtcagaagt
     5221 attttaactc atccaaaagc cccagacacc gcattatttc ccggtgcatg cattcttctc
     5281 tgacaagtac cttctctctc cattggctgg ataaaagaat ctgtaggtcg ccgacatctt
     5341 gtttgtgtct acatggccaa catacttctc ttacaatgta tgtgcatatg catatgtaag
     5401 tggagcgtgg tgggaggctt tattgtagag ctattgttct gacggagtag gtgcaaacgg
     5461 agtctgttta ggtacagtca ccatctctgc gtatctgtac attttcactg gagctagaag
     5521 gagctactct tcgacttcga gaacagggga gcaacccagt ttgtgttcaa ggcgattgtt
     5581 agttgctccg gcaagataag gttacattat tctctacgct ttagcgttta ggggtactcg
     5641 gataaatgcc agatgaaaaa cgctaggtgc atacagtaca agtcgagccg acattgttgg
     5701 accgccccga taattattgt atgttgtgtt tgcttgtaag accatgattg atatttattg
     5761 ggtggttcag caatcggact cgttgattat tattattatt gaccctcatg atatacaatg
     5821 gattttattt ctgagcaatc gtagcaaaat attcgacgca atgtagcttg agtttatgac
     5881 cttagcggac atcaccagcg cagctttaca ttttccattc tttggattta tttttcttat
     5941 ccactaggag attagaagtc taagtatcat gtaagaaagg agataattgt tgtctgctgg
     6001 ataataaaat tttgctgagt tagaaatgag atacgaatca gcaagatgtc tttggttgca
     6061 gcctgtagag atttagattt cgacaatctg actaaaatgt acgaagagaa aaatagacac
     6121 atttagtcaa tacttcagcg ttaagggtgc tatagactgt gcaacttcgt ttcactgctg
     6181 acatgaaatt ccagagactt aacatattac atgattataa ataatttgcg tataatatgt
     6241 ttggcataaa gcgatttctg gaaaagttgc gaaaaattcc gaattactac aacttaccaa
     6301 aaaaacagag gcatattttg ctaaatcacc atatcctata cwgagctcma kcgsaaagaa
     6361 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     6421 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     6481 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     6541 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6601 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6661 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6721 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6781 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     6841 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     6901 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6961 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     7021 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     7081 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     7141 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     7201 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     7261 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     7321 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     7381 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     7441 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     7501 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     7561 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     7621 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     7681 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     7741 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     7801 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     7861 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     7921 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     7981 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     8041 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     8101 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     8161 agtgccacct gacgtc
//
