LOCUS       sequence-417329-        8320 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 8320)
  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 8320)
  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: pWR54
FEATURES             Location/Qualifiers
     source          1..8320
                     /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             3197..4825
                     /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             4147..4155
                     /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(6565..7153)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6645..6664)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7324..8115,8116..8184))
                     /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     7947..7966
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8185..8289)
                     /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 tggatctcac aaagaaccaa caaactatgc attattcatt agccatggcc
     2341 ataatacatg gctgatggtt tcatctctat atttgttcta tatttaggcg tttgtgaatg
     2401 atatatacta atagacaata gaatatgacg acagaccatt tccaacaagg cgcaaagcac
     2461 caaggctcca aacgtgtgtg gcataaggtc ttccggaatg caccagatgc tgaagacttt
     2521 aatgtggcat ttttctttca aacttgagca acaagtttgc gtctgccaga acacgtaaga
     2581 aacgaataat ggtaatagat gagtctaccg tgtttgctag tatttacgct gtactattta
     2641 ctaataagta tcaggggctc ctcttgtgtc tagaagttat gaatgagttc ccatggctgt
     2701 gtgcctagat gcgctaccgc gttcagcctg aaattggtac tctaaaccta atacacgcct
     2761 ctcgagttta gacaaggtta tatttaatgg attcgcgcgt atgcctcaaa tttttttgat
     2821 ttgcattttt tgtttacgcg tttgttttga attcggtttg tttaccaaac agtgcgtacc
     2881 ttaatttttc tgatttgcgt agagatcgcc gagcacatag cgactcctta ccaggctcta
     2941 gaattatagc gtggatcctt cttatggtta tggactatta ttattttgat ttatggaagg
     3001 gggtctcgct tattgtcgtt tgccgtagca gggcacgtca aacgcgtcat aaaccagaat
     3061 ggttacaata tcgagagttg ggcagcgcca actgtctgcc tttgaggcca cctcaaacca
     3121 cactataaat aagggcagat gtaccctcaa tgcccaaaag atgaacactc tacttcttaa
     3181 ctcaaactag ggtaccatgg tgaagcgtga gaaaaatgtc atctatggcc ctgagcctct
     3241 ccatcctttg gaggatttga ctgccggcga aatgttgttt cgtgctctcc gcaagcactc
     3301 tcatttgcct caagccttgg tcgatgtggt cggcgatgaa tctttgagct acaaggagtt
     3361 ttttgaggca accgtcttgt tggctcagtc cctccacaat tgtggctaca agatgaacga
     3421 cgtcgttagt atctgtgctg aaaacaatac ccgtttcttc attccagtca tcgccgcatg
     3481 gtatatcggt atgatcgtgg ctccagtcaa cgagagctac attcccgacg aattgtgtaa
     3541 agtcatgggt atctctaagc cacagattgt cttcaccact aagaatattt tgaacaaagt
     3601 cttggaagtc caaagccgca ccaactttat taagcgtatc atcatcttgg acactgtgga
     3661 gaatattcac ggttgcgaat ctttgcctaa tttcatctct cgctattcag acggcaacat
     3721 cgcaaacttt aaaccactcc acttcgaccc tgtggaacaa gttgcagcca ttttgtgtag
     3781 cagcggtact actggactcc caaagggagt catgcagacc catcaaaaca tttgcgtgcg
     3841 tttgatccat gctctcgatc cacgtgtggg cactcagttg attcctggtg tcaccgtctt
     3901 ggtctacttg cctttcttcc atgctttcgg ctttagcatt actttgggtt actttatggt
     3961 cggtctccgc gtgattatgt tccgccgttt tgatcaggag gctttcttga aagccatcca
     4021 agattatgaa gtccgcagtg tcatcaacgt gcctagcgtg atcttgtttt tgtctaagag
     4081 cccactcgtg gacaagtacg acttgtcttc attgcgtgaa ttgtgttgcg gtgccgctcc
     4141 attggctaag gaggtcgctg aagtggccgc caaacgcttg aatcttccag gtattcgttg
     4201 tggcttcggc ctcaccgaat ctaccagcgc taacattcac tctctcggtg atgagtttaa
     4261 gagcggctct ttgggccgtg tcactccact catggctgct aagatcgctg atcgcgaaac
     4321 tggtaaggct ttgggtccga accaagtggg cgagttgtgt atcaaaggcc ctatggtgag
     4381 caagggttat gtcaataacg ttgaagctac caaggaggcc atcgacgacg acggctggtt
     4441 gcattctggt gattttggat attacgacga agatgagcat ttttacgtcg tggatcgtta
     4501 caaggagttg atcaaataca agggtagcca ggttgctcca gctgagttgg aggagatttt
     4561 gttgaaaaat ccatgcattc gcgatgtcgc tgtggtcggc attcctgatt tggaggccgg
     4621 cgaattgcct tctgctttcg ttgtcaagca gcctggtaaa gaaattaccg ccaaagaagt
     4681 gtatgattac ttggctgaac gtgtgagcca tactaagtac ttgcgtggcg gcgtgcgttt
     4741 tgttgactcc atccctcgta acgtaacagg caaaattacc cgcaaggagt tgttgaaaca
     4801 attgttggag aaggccggcg gttagcttaa gcattttaga caagtgccta tttattcaga
     4861 ttgatttttt atattgaata tggatgttga ttacgacttg tgttacgaag catgaacatg
     4921 tgatttgaaa aagtgtttac gaataaaaga actttttttg caaccgttgc tagttgaagt
     4981 gtagactctg aatgcgaagg gttgcaaatg tgcccagtag aaaaaatatt ccatattttt
     5041 ctgaacttaa ttcaatgaaa tacgagtatt actacaactg ccaatatcgg aacaatttaa
     5101 cgagagcaag ctatataaaa tgaatgttca tttacacaag ctagaaccaa aacttgaatt
     5161 aaaatttaac ataactaaat gtaactagat aaaagagaga cataaccaaa cgttcagaag
     5221 gcagaagaat tcagttaact cctgcaggaa tcgattggcc ggcctcacgt ggacgcgtta
     5281 ttaatcgttt aaacctacgt aaccgcggct taattaaagc tagccagcgc ttgagctctc
     5341 tgcggtgtct acaaggtcag aagtatttta actcatccaa aagccccaga caccgcatta
     5401 tttcccggtg catgcattct tctctgacaa gtaccttctc tctccattgg ctggataaaa
     5461 gaatctgtag gtcgccgaca tcttgtttgt gtctacatgg ccaacatact tctcttacaa
     5521 tgtatgtgca tatgcatatg taagtggagc gtggtgggag gctttattgt agagctattg
     5581 ttctgacgga gtaggtgcaa acggagtctg tttaggtaca gtcaccatct ctgcgtatct
     5641 gtacattttc actggagcta gaaggagcta ctcttcgact tcgagaacag gggagcaacc
     5701 cagtttgtgt tcaaggcgat tgttagttgc tccggcaaga taaggttaca ttattctcta
     5761 cgctttagcg tttaggggta ctcggataaa tgccagatga aaaacgctag gtgcatacag
     5821 tacaagtcga gccgacattg ttggaccgcc ccgataatta ttgtatgttg tgtttgcttg
     5881 taagaccatg attgatattt attgggtggt tcagcaatcg gactcgttga ttattattat
     5941 tattgaccct catgatatac aatggatttt atttctgagc aatcgtagca aaatattcga
     6001 cgcaatgtag cttgagttta tgaccttagc ggacatcacc agcgcagctt tacattttcc
     6061 attctttgga tttatttttc ttatccacta ggagattaga agtctaagta tcatgtaaga
     6121 aaggagataa ttgttgtctg ctggataata aaattttgct gagttagaaa tgagatacga
     6181 atcagcaaga tgtctttggt tgcagcctgt agagatttag atttcgacaa tctgactaaa
     6241 atgtacgaag agaaaaatag acacatttag tcaatacttc agcgttaagg gtgctataga
     6301 ctgtgcaact tcgtttcact gctgacatga aattccagag acttaacata ttacatgatt
     6361 ataaataatt tgcgtataat atgtttggca taaagcgatt tctggaaaag ttgcgaaaaa
     6421 ttccgaatta ctacaactta ccaaaaaaac agaggcatat tttgctaaat caccatatcc
     6481 tatactgagc tccatcggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     6541 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     6601 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     6661 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     6721 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     6781 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     6841 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     6901 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     6961 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     7021 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     7081 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     7141 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     7201 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     7261 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     7321 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     7381 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     7441 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     7501 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     7561 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     7621 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     7681 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     7741 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     7801 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     7861 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     7921 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     7981 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     8041 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     8101 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     8161 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     8221 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     8281 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
