LOCUS       sequence-409762-        7723 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 7723)
  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 7723)
  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: pWR24
FEATURES             Location/Qualifiers
     source          1..7723
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2600..4228
                     /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             3550..3558
                     /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(5968..6556)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6048..6067)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6727..7518,7519..7587))
                     /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     7350..7369
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7588..7692)
                     /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 gtactactct tgacgacacg gcttaccgtt acagaaccag tgtcccaggg gacgccgagg
      841 ccatcgaggc attggatggg tccttcacca ccgacaccgt cttcagagtc accgccaccg
      901 gggacggctt caccttgaga gaggtgccgg tggacccgcc cttgaccaag gtgttccccg
      961 acgacgaatc ggacgacgaa tcggacgacg gggaggacgg cgacccggac tccagaacgt
     1021 tcgtcgcgta cggggacgac ggcgacttgg cgggcttcgt ggtcatctcg tactcggcgt
     1081 ggaacagaag acttaccgtc gaggacatcg aggtcgcccc ggagcacaga gggcacgggg
     1141 tcgggagagc gttgatgggg ctcgcgacgg agttcgccgg cgagagaggc gccgggcacc
     1201 tctggttgga ggtcaccaac gtcaacgcac cggcgatcca cgcttacaga agaatggggt
     1261 tcaccctctg cggcttggac accgcccttt acgacggcac cgcctcggac ggcgagagac
     1321 aggcgctcta catgagcatg ccctgcccct aagcggccgc taaccacttg caaaatcctt
     1381 tgtattcttg ttctgcaaac attttgtcca atctcttatc tttctacgat gttgaagaac
     1441 atacattttt gttagtccag cttgttatcc tttttttatt aaataaaaaa ttttattgtt
     1501 aaagttgttt ttcttctttg tctgtcctgt tttaactcat atatattcta tatattacag
     1561 aaagaaagta gatcatctaa aataaacatt tatttgatga gacaaagtag gttttttaga
     1621 atacaatgta gcatcctcta atatacaata gagttgtttt ataaaagcga tacatttaat
     1681 attatgacag gactggtgaa gtgtatagaa ttgtgctaat ctggcaaacg aacccaagaa
     1741 gacgttaaag caaatagtgc attttgtatt acaatcaagt tcgtcgacca tgcattagat
     1801 cttgcatgcc tctagaacct aggtggcgcc acagctgtgg atcctcgtta gtatttccgt
     1861 gaaggcagcc attgtgaaaa ttgaacatca cctgaggtat tttagccacc agaagcggcg
     1921 gtacggaaga aagtgtgtac gatggttggt ggtggaattg cgtgcatgcc tgatggggca
     1981 atattaatta gatagagctt tggtgatatt agtggataat agaattcaca gagaagacat
     2041 caggagcaat ttccaagagc cattgatgat gtaattgccc caacagcaag attcagatct
     2101 gacaattgac caccgttttg tagaagcaaa aaatcgtaga ttatcaccaa gagggttttt
     2161 caccgaacca gcaaatagaa actatgccgt agaactcgcc caggcttttt tgctagcact
     2221 ttccagcagt agaaccgtcc aattaagtca acaggaacca ttgaggtcga gcccaaccac
     2281 ctgaaccccc tcacggtcgt gtccctatta ttgatccaga gggtgccagt ttcggtagcc
     2341 aatattggtt catgggtttc tatggcccgg agtgagtttg caggttggcc ccggcgccgt
     2401 ctgcaggatg ggagttatag cggccaaact tcacatttcg aaatcctgct gcagccaatc
     2461 tgaagaatta atataaattc gtgtcgaatc gccgtctgtg aaatttcagt acttgatttt
     2521 cttttcttct tctttttctc ttttgtttct tcagaatcaa tacacatttt ttcttcccta
     2581 taaacaattc atcggtacca tggtgaagcg tgagaaaaat gtcatctatg gccctgagcc
     2641 tctccatcct ttggaggatt tgactgccgg cgaaatgttg tttcgtgctc tccgcaagca
     2701 ctctcatttg cctcaagcct tggtcgatgt ggtcggcgat gaatctttga gctacaagga
     2761 gttttttgag gcaaccgtct tgttggctca gtccctccac aattgtggct acaagatgaa
     2821 cgacgtcgtt agtatctgtg ctgaaaacaa tacccgtttc ttcattccag tcatcgccgc
     2881 atggtatatc ggtatgatcg tggctccagt caacgagagc tacattcccg acgaattgtg
     2941 taaagtcatg ggtatctcta agccacagat tgtcttcacc actaagaata ttttgaacaa
     3001 agtcttggaa gtccaaagcc gcaccaactt tattaagcgt atcatcatct tggacactgt
     3061 ggagaatatt cacggttgcg aatctttgcc taatttcatc tctcgctatt cagacggcaa
     3121 catcgcaaac tttaaaccac tccacttcga ccctgtggaa caagttgcag ccattttgtg
     3181 tagcagcggt actactggac tcccaaaggg agtcatgcag acccatcaaa acatttgcgt
     3241 gcgtttgatc catgctctcg atccacgtgt gggcactcag ttgattcctg gtgtcaccgt
     3301 cttggtctac ttgcctttct tccatgcttt cggctttagc attactttgg gttactttat
     3361 ggtcggtctc cgcgtgatta tgttccgccg ttttgatcag gaggctttct tgaaagccat
     3421 ccaagattat gaagtccgca gtgtcatcaa cgtgcctagc gtgatcttgt ttttgtctaa
     3481 gagcccactc gtggacaagt acgacttgtc ttcattgcgt gaattgtgtt gcggtgccgc
     3541 tccattggct aaggaggtcg ctgaagtggc cgccaaacgc ttgaatcttc caggtattcg
     3601 ttgtggcttc ggcctcaccg aatctaccag cgctaacatt cactctctcg gtgatgagtt
     3661 taagagcggc tctttgggcc gtgtcactcc actcatggct gctaagatcg ctgatcgcga
     3721 aactggtaag gctttgggtc cgaaccaagt gggcgagttg tgtatcaaag gccctatggt
     3781 gagcaagggt tatgtcaata acgttgaagc taccaaggag gccatcgacg acgacggctg
     3841 gttgcattct ggtgattttg gatattacga cgaagatgag catttttacg tcgtggatcg
     3901 ttacaaggag ttgatcaaat acaagggtag ccaggttgct ccagctgagt tggaggagat
     3961 tttgttgaaa aatccatgca ttcgcgatgt cgctgtggtc ggcattcctg atttggaggc
     4021 cggcgaattg ccttctgctt tcgttgtcaa gcagcctggt aaagaaatta ccgccaaaga
     4081 agtgtatgat tacttggctg aacgtgtgag ccatactaag tacttgcgtg gcggcgtgcg
     4141 ttttgttgac tccatccctc gtaacgtaac aggcaaaatt acccgcaagg agttgttgaa
     4201 acaattgttg gagaaggccg gcggttagct taagcatttt agacaagtgc ctatttattc
     4261 agattgattt tttatattga atatggatgt tgattacgac ttgtgttacg aagcatgaac
     4321 atgtgatttg aaaaagtgtt tacgaataaa agaacttttt ttgcaaccgt tgctagttga
     4381 agtgtagact ctgaatgcga agggttgcaa atgtgcccag tagaaaaaat attccatatt
     4441 tttctgaact taattcaatg aaatacgagt attactacaa ctgccaatat cggaacaatt
     4501 taacgagagc aagctatata aaatgaatgt tcatttacac aagctagaac caaaacttga
     4561 attaaaattt aacataacta aatgtaacta gataaaagag agacataacc aaacgttcag
     4621 aaggcagaag aattcagtta actcctgcag gaatcgattg gccggcctca cgtggacgcg
     4681 ttattaatcg tttaaaccta cgtaaccgcg gcttaattaa agctagccag cgcttgagct
     4741 cwstryrgkr tctacaaggt cagaagtatt ttaactcatc caaaagcccc agacaccgca
     4801 ttatttcccg gtgcatgcat tcttctctga caagtacctt ctctctccat tggctggata
     4861 aaagaatctg taggtcgccg acatcttgtt tgtgtctaca tggccaacat acttctctta
     4921 caatgtatgt gcatatgcat atgtaagtgg agcgtggtgg gaggctttat tgtagagcta
     4981 ttgttctgac ggagtaggtg caaacggagt ctgtttaggt acagtcacca tctctgcgta
     5041 tctgtacatt ttcactggag ctagaaggag ctactcttcg acttcgagaa caggggagca
     5101 acccagtttg tgttcaaggc gattgttagt tgctccggca agataaggtt acattattct
     5161 ctacgcttta gcgtttaggg gtactcggat aaatgccaga tgaaaaacgc taggtgcata
     5221 cagtacaagt cgagccgaca ttgttggacc gccccgataa ttattgtatg ttgtgtttgc
     5281 ttgtaagacc atgattgata tttattgggt ggttcagcaa tcggactcgt tgattattat
     5341 tattattgac cctcatgata tacaatggat tttatttctg agcaatcgta gcaaaatatt
     5401 cgacgcaatg tagcttgagt ttatgacctt agcggacatc accagcgcag ctttacattt
     5461 tccattcttt ggatttattt ttcttatcca ctaggagatt agaagtctaa gtatcatgta
     5521 agaaaggaga taattgttgt ctgctggata ataaaatttt gctgagttag aaatgagata
     5581 cgaatcagca agatgtcttt ggttgcagcc tgtagagatt tagatttcga caatctgact
     5641 aaaatgtacg aagagaaaaa tagacacatt tagtcaatac ttcagcgtta agggtgctat
     5701 agactgtgca acttcgtttc actgctgaca tgaaattcca gagacttaac atattacatg
     5761 attataaata atttgcgtat aatatgtttg gcataaagcg atttctggaa aagttgcgaa
     5821 aaattccgaa ttactacaac ttaccaaaaa aacagaggca tattttgcta aatcaccata
     5881 tcctatacwg agctcmakcg swaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5941 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     6001 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     6061 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     6121 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     6181 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     6241 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     6301 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     6361 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     6421 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6481 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6541 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     6601 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6661 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6721 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6781 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6841 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     6901 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6961 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     7021 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     7081 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     7141 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     7201 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     7261 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     7321 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     7381 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     7441 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     7501 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     7561 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     7621 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     7681 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
