LOCUS       sequence-409745-        9782 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  integrating bioluminescent reporter plasmid for S. stipitis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9782)
  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 9782)
  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: pWR16
FEATURES             Location/Qualifiers
     source          1..9782
                     /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(8027..8615)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8107..8126)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8786..9577,9578..9646))
                     /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     9409..9428
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9647..9751)
                     /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 gctcagtacg aacaagagaa tgaaaagctc
     5221 ggttcgaatc aagacataac ttacttcaag gacttggtgg aactttccaa gaaatcaata
     5281 tcattgactt ctgaattatc tgctgtgata aagagaccag aattcaaggg caaggagttg
     5341 acctacacat tgattcaagc attggcacac cagtgtttca atccatgtcg cgatgttcgt
     5401 tcctttgctt tgaacccctt gcaagcaatg gttttatcta ttgacatcaa cgaagagtta
     5461 actgcatttg gaatattcga gtatggattg tttccattgt tatcggaatt gtccaagagt
     5521 gaagttttgc aaaccgatat tcacggtttt gtcaagacgc agactgaaac attgaccttg
     5581 gtcagcaagg tttatcttca ctactgtgtc gacttgaagc aggaagaaac tgacggtgtt
     5641 tggttaggaa tattggataa ctttattaca ttccacgacc aagaaagcaa gctcaagaaa
     5701 gagaattcgt tcaaagaagc aggagccgaa ttactcaaaa acatgattct agtactacaa
     5761 gacaatggta ctttgagcaa ggacaaagag atcctctgga acgaatcatg gctgaagata
     5821 gataagattt atcccggttt aagagacgaa ttggatccgg agccgttgtc tgagaagaat
     5881 aaagagaata tagtcgacac ggctgctgag ggtggagaag agaagaaaat cgaaacagac
     5941 ggagataact aatctgcaat aaatattaca cttatgtaat ctaatggttt cttaagaaag
     6001 tagtcgttat ggagaagtag tattttgtat tttagttact ttcataaata tagcaagctt
     6061 atgttgcatc tcaaacagtg cgaaaaatat tttagtctat tttatagtat ctacaaacaa
     6121 attttcaatt catcacagac tttctttgtg tactagcaac accctaaaaa cattcgacaa
     6181 tggtcaacgt ccaaacctat acacaaaggg ccgagtctca ccagtcaccg gtagcacagc
     6241 gtctcttcaa attaatggac agcaagaaaa ctaacttgtg tgcttcagtg gatgtgaaaa
     6301 caacctctga gttcttatct ttgattgata agttgggtcc ttacatttgt ttggttaaga
     6361 cccatatcga cattatcgac gatttttcgt atgaaggtac cattgttcct ttgttggaat
     6421 tgtccaagaa gcacaacttc atgatctttg aagatcgtaa attcgcagac attggaaaca
     6481 ctgtcaagtc tcagtattca ggtggagtct acaagattgc ccaatggtca gatatcacca
     6541 atgctcacgg tgttactggt tctggtattg ttcagggatt aaaggaagct gcaagagaaa
     6601 ctaccgacga accaagaggt ttattgatgc ttgccgaact atcttctaag ggctcgattg
     6661 ctcatggtaa gtatactgaa gaaactgtgg aaatcgccaa aactgacaaa gagtttgtta
     6721 ttgggttcat tgcccaaaga gacatgggtg gacaagatga aggtttcgat tggattgtta
     6781 tgactcctgg tgtcggtttg gatgacaagg gtgacagttt aggtcaacaa tatagaacag
     6841 tagatgaagt tgtttccaca ggaaccgata ttatcattgt tggtagaggc ttattcggca
     6901 agggtagaga tcctgatgta gagggccaaa gatacagaga agctggatgg aacgcttact
     6961 tgaagaagac aagccaagtg taaaatatgt aaaattttgt aaaaatcgta aaactgtaat
     7021 gtacatcgtt aagtatttac aaacaactat atgattattt acaagcagag gcctattcca
     7081 aaatttcgaa ttccagtaag tcgtcagtca atctggttct cattatttca aaaaccaaga
     7141 ggggcacgga tgtagccggt attgttgtca atgcattgcg gacaaaacct ttgtatgcca
     7201 tttgatacca agtcaacttt gattttgtct tcatcctaag gatttggtta tatgtatcaa
     7261 tgtatgagtt gtagtacaag gttatggagg gtttgtatga acgcgtagct gatgcattgt
     7321 agaaatcaag agcttcaagt ctagaaagat gaatcttctg aatcttggtg attgggtatt
     7381 gtattgccaa caacgagaaa gcggcagatg caccagctac gagaataaag gttgacctca
     7441 atatcttttt ggttctcgta cgctccaagc tcaatagtcg ttcgtctatc tgttcttcgt
     7501 tcatatttga gtattgtcgt aacttgcttt tgatggattc tttgcttcgt ctataaacat
     7561 tgacaacctt gaaagttgct gtatagcctt gagtcttgac gaactcaaag gtagagaagt
     7621 agaaggcaaa tccaagcgat tcttttacaa gcgaaaaact atacccagca aaaactccaa
     7681 ccaatccgac ttctttaagt ttgttcaaac cgtacatcca gaggttttgg tgtgaaccac
     7741 tcaacatttc tgctgctgtc agtctagtat atatggcgtc tacaggtgct gctgcgagtg
     7801 actgaacggc accagcaata aaaccagctc tccatgtatc gaatggggag taatagaaca
     7861 attctcgggg tttatctgct tttggggtat gcagactgtt gaatcgatcc aaagccgtca
     7921 agtaagttcc gtacaatatc aaccctgtga gctccatcgg aaagaacatg tgagcaaaag
     7981 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     8041 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     8101 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     8161 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     8221 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     8281 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     8341 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     8401 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     8461 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     8521 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     8581 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     8641 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     8701 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     8761 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     8821 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     8881 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     8941 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     9001 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     9061 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     9121 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     9181 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     9241 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     9301 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     9361 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     9421 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     9481 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     9541 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     9601 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     9661 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     9721 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     9781 tc
//
