LOCUS       sequence-455380-        9755 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  integrating bioluminescene reporter for Cas9 activity in S. 
            stipitis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9755)
  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 9755)
  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: pWR58
FEATURES             Location/Qualifiers
     source          1..9755
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1721..1729
                     /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)"
     RBS             3828..3836
                     /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(8000..8588)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8080..8099)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8759..9550,9551..9619))
                     /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     9382..9401
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9620..9724)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccaatgatac cggtctcgtt agtatttccg tgaaggcagc cattgtgaaa attgaacatc
       61 acctgaggta ttttagccac cagaagcggc ggtacggaag aaagtgtgta cgatggttgg
      121 tggtggaatt gcgtgcatgc ctgatggggc aatattaatt agatagagct ttggtgatat
      181 tagtggataa tagaattcac agagaagaca tcaggagcaa tttccaagag ccattgatga
      241 tgtaattgcc ccaacagcaa gattcagatc tgacaattga ccaccgtttt gtagaagcaa
      301 aaaatcgtag attatcacca agagggtttt tcaccgaacc agcaaataga aactatgccg
      361 tagaactcgc ccaggctttt ttgctagcac tttccagcag tagaaccgtc caattaagtc
      421 aacaggaacc attgaggtcg agcccaacca cctgaacccc ctcacggtcg tgtccctatt
      481 attgatccag agggtgccag tttcggtagc caatattggt tcatgggttt ctatggcccg
      541 gagtgagttt gcaggttggc cccggcgccg tctgcaggat gggagttata gcggccaaac
      601 ttcacatttc gaaatcctgc tgcagccaat ctgaagaatt aatataaatt cgtgtcgaat
      661 cgccgtctgt gaaatttcag tacttgattt tcttttcttc ttctttttct cttttgtttc
      721 ttcagaatca atacacattt tttcttccct ataaacaatt catcggtacc atggtgaagc
      781 gtgagaaaaa tgtcatctat ggccctgagc ctctccatcc tttggaggat ttgactgccg
      841 gcgaaatgtt gtttcgtgct ctccgcaagc actctcattt gcctcaagcc ttggtcgatg
      901 tggtcggcga tgaatctttg agctacaagg agttttttga ggcaaccgtc ttgttggctc
      961 agtccctcca caattgtggc tacaagatga acgacgtcgt tagtatctgt gctgaaaaca
     1021 atacccgttt cttcattcca gtcatcgccg catggtatat cggtatgatc gtggctccag
     1081 tcaacgagag ctacattccc gacgaattgt gtaaagtcat gggtatctct aagccacaga
     1141 ttgtcttcac cactaagaat attttgaaca aagtcttgga agtccaaagc cgcaccaact
     1201 ttattaagcg tatcatcatc ttggacactg tggagaatat tcacggttgc gaatctttgc
     1261 ctaatttcat ctctcgctat tcagacggca acatcgcaaa ctttaaacca ctccacttcg
     1321 accctgtgga acaagttgca gccattttgt gtagcagcgg tactactgga ctcccaaagg
     1381 gagtcatgca gacccatcaa aacatttgcg tgcgtttgat ccatgctctc gatccacgtg
     1441 tgggcactca gttgattcct ggtgtcaccg tcttggtcta cttgcctttc ttccatgctt
     1501 tcggctttag cattactttg ggttacttta tggtcggtct ccgcgtgatt atgttccgcc
     1561 gttttgatca ggaggctttc ttgaaagcca tccaagatta tgaagtccgc agtgtcatca
     1621 acgtgcctag cgtgatcttg tttttgtcta agagcccact cgtggacaag tacgacttgt
     1681 cttcattgcg tgaattgtgt tgcggtgccg ctccattggc taaggaggtc gctgaagtgg
     1741 ccgccaaacg cttgaatctt ccaggtattc gttgtggctt cggcctcacc gaatctacca
     1801 gcgctaacat tcactctctc ggtgatgagt ttaagagcgg ctctttgaaa gttctactta
     1861 gtccccgggg ctcgagccgt acacttattg ttaactatga aacatatact tatggacaag
     1921 gttcactgat ttggactgaa aagcaaaaac gagaaatgag tcaacgtctg agatcaatcc
     1981 ctagaccacc cgggaaggtc atgtgtttca tgaagtacga taaggttggt aaccgattga
     2041 ctcattggtt cgtggcggag aagtacgcag agtaaaaccg gggccgattc gtggtaaatt
     2101 ctggaatgat ccagaggcgc gacatttatg cagacaattt gtgttttgtc gcaaacgatg
     2161 ttatagcgaa atttttcact ctgtcagata aatggatttt gtcaaaaggg ggaagtagaa
     2221 ggagaatggg cccgagatgt tctgccaaat tctcagtagc ataatgtgaa agaagccctt
     2281 acattgtcca gcctctggca tcattaaaaa ccgtagcgga aaccaattgt ctctgttctt
     2341 ccctggcaca ccctggtagc cccacccagt tgtagtacat ctcacacgct ggcaacttgg
     2401 gacaatcagc aacttttttt tcttttaatt ttttcagcgc gacattttgc ctcttctgcg
     2461 agaacagact ttttcacctc catctcaccc ccctttgcac ttatataaat tggaccagtt
     2521 cctcccattg tagaaaaaat tttgctggac ctttttctct tttttttgtc ctttagtttc
     2581 atacaatcta agtctatcta catgtacaat ggccaagttg accagtgccg ttccagtgct
     2641 caccgctcgc gacgtcgccg gagctgtcga gttctggacc gaccggctcg gtttctccag
     2701 agacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgacct tgttcatcag
     2761 cgctgtccag gaccaggtgg tgccagacaa caccttggcc tgggtgtggg tgcgcggctt
     2821 ggacgagttg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg
     2881 tccagccatg accgagatcg gcgagcagcc atggggtcgg gagttcgcct tgcgcgaccc
     2941 agccggcaac tgcgtgcact tcgtggccga ggagcaggac tgagcggccg ctaaccactt
     3001 gcaaaatcct ttgtattctt gttctgcaaa cattttgtcc aatctcttat ctttctacga
     3061 tgttgaagaa catacatttt tgttagtcca gcttgttatc ctttttttat taaataaaaa
     3121 attttattgt taaagttgtt tttcttcttt gtctgtcctg ttttaactca tatatattct
     3181 atatattaca gaaagaaagt agatcatcta aaataaacat ttatttgatg agacaaagta
     3241 ggttttttag aatacaatgt agcatcctct aatatacaat agagttgttt tataaaagcg
     3301 atacatttaa tattatgaca ggactggtga agtgtataga attgtgctaa tctggcaaac
     3361 gaacccaaga agacgttaaa gcaaatagtg cattttgtat tacaatcaag ttcgtcgaca
     3421 aagttctact tagtccccgg ggttgcagcc attttgtgta gcagcggtac tactggactc
     3481 ccaaagggag tcatgcagac ccatcaaaac atttgcgtgc gtttgatcca tgctctcgat
     3541 ccacgtgtgg gcactcagtt gattcctggt gtcaccgtct tggtctactt gcctttcttc
     3601 catgctttcg gctttagcat tactttgggt tactttatgg tcggtctccg cgtgattatg
     3661 ttccgccgtt ttgatcagga ggctttcttg aaagccatcc aagattatga agtccgcagt
     3721 gtcatcaacg tgcctagcgt gatcttgttt ttgtctaaga gcccactcgt ggacaagtac
     3781 gacttgtctt cattgcgtga attgtgttgc ggtgccgctc cattggctaa ggaggtcgct
     3841 gaagtggccg ccaaacgctt gaatcttcca ggtattcgtt gtggcttcgg cctcaccgaa
     3901 tctaccagcg ctaacattca ctctctcggt gatgagttta agagcggctc tttgggccgt
     3961 gtcactccac tcatggctgc taagatcgct gatcgcgaaa ctggtaaggc tttgggtccg
     4021 aaccaagtgg gcgagttgtg tatcaaaggc cctatggtga gcaagggtta tgtcaataac
     4081 gttgaagcta ccaaggaggc catcgacgac gacggctggt tgcattctgg tgattttgga
     4141 tattacgacg aagatgagca tttttacgtc gtggatcgtt acaaggagtt gatcaaatac
     4201 aagggtagcc aggttgctcc agctgagttg gaggagattt tgttgaaaaa tccatgcatt
     4261 cgcgatgtcg ctgtggtcgg cattcctgat ttggaggccg gcgaattgcc ttctgctttc
     4321 gttgtcaagc agcctggtaa agaaattacc gccaaagaag tgtatgatta cttggctgaa
     4381 cgtgtgagcc atactaagta cttgcgtggc ggcgtgcgtt ttgttgactc catccctcgt
     4441 aacgtaacag gcaaaattac ccgcaaggag ttgttgaaac aattgttgga gaaggccggc
     4501 ggttagctta agcattttag acaagtgcct atttattcag attgattttt tatattgaat
     4561 atggatgttg attacgactt gtgttacgaa gcatgaacat gtgatttgaa aaagtgttta
     4621 cgaataaaag aacttttttt gcaaccgttg ctagttgaag tgtagactct gaatgcgaag
     4681 ggttgcaaat gtgcccagta gaaaaaatat tccatatttt tctgaactta attcaatgaa
     4741 atacgagtat tactacaact gccaatatcg gaacaattta acgagagcaa gctatataaa
     4801 atgaatgttc atttacacaa gctagaacca aaacttgaat taaaatttaa cataactaaa
     4861 tgtaactaga taaaagagag acataaccaa acgttcagaa ggcagaatct agaacctagg
     4921 tggcgccaca gctgtggatc ccgtgcacca agcttgttat aaaggcgcgc ctcaattgga
     4981 ctagttttag gcctttggac gtcctgatca aggtaccacc atggacttaa ggtggccaac
     5041 tcatatggcg tacgagaatt cagttaactc ctgcaggaat cgattggccg gcctcacgtg
     5101 gacgcgttat taatcgttta aacctacgta accgcggctt aattaaagct agccagcgct
     5161 tgagctcagt acgaacaaga gaatgaaaag ctcggttcga atcaagacat aacttacttc
     5221 aaggacttgg tggaactttc caagaaatca atatcattga cttctgaatt atctgctgtg
     5281 ataaagagac cagaattcaa gggcaaggag ttgacctaca cattgattca agcattggca
     5341 caccagtgtt tcaatccatg tcgcgatgtt cgttcctttg ctttgaaccc cttgcaagca
     5401 atggttttat ctattgacat caacgaagag ttaactgcat ttggaatatt cgagtatgga
     5461 ttgtttccat tgttatcgga attgtccaag agtgaagttt tgcaaaccga tattcacggt
     5521 tttgtcaaga cgcagactga aacattgacc ttggtcagca aggtttatct tcactactgt
     5581 gtcgacttga agcaggaaga aactgacggt gtttggttag gaatattgga taactttatt
     5641 acattccacg accaagaaag caagctcaag aaagagaatt cgttcaaaga agcaggagcc
     5701 gaattactca aaaacatgat tctagtacta caagacaatg gtactttgag caaggacaaa
     5761 gagatcctct ggaacgaatc atggctgaag atagataaga tttatcccgg tttaagagac
     5821 gaattggatc cggagccgtt gtctgagaag aataaagaga atatagtcga cacggctgct
     5881 gagggtggag aagagaagaa aatcgaaaca gacggagata actaatctgc aataaatatt
     5941 acacttatgt aatctaatgg tttcttaaga aagtagtcgt tatggagaag tagtattttg
     6001 tattttagtt actttcataa atatagcaag cttatgttgc atctcaaaca gtgcgaaaaa
     6061 tattttagtc tattttatag tatctacaaa caaattttca attcatcaca gactttcttt
     6121 gtgtactagc aacaccctaa aaacattcga caatggtcaa cgtccaaacc tatacacaaa
     6181 gggccgagtc tcaccagtca ccggtagcac agcgtctctt caaattaatg gacagcaaga
     6241 aaactaactt gtgtgcttca gtggatgtga aaacaacctc tgagttctta tctttgattg
     6301 ataagttggg tccttacatt tgtttggtta agacccatat cgacattatc gacgattttt
     6361 cgtatgaagg taccattgtt cctttgttgg aattgtccaa gaagcacaac ttcatgatct
     6421 ttgaagatcg taaattcgca gacattggaa acactgtcaa gtctcagtat tcaggtggag
     6481 tctacaagat tgcccaatgg tcagatatca ccaatgctca cggtgttact ggttctggta
     6541 ttgttcaggg attaaaggaa gctgcaagag aaactaccga cgaaccaaga ggtttattga
     6601 tgcttgccga actatcttct aagggctcga ttgctcatgg taagtatact gaagaaactg
     6661 tggaaatcgc caaaactgac aaagagtttg ttattgggtt cattgcccaa agagacatgg
     6721 gtggacaaga tgaaggtttc gattggattg ttatgactcc tggtgtcggt ttggatgaca
     6781 agggtgacag tttaggtcaa caatatagaa cagtagatga agttgtttcc acaggaaccg
     6841 atattatcat tgttggtaga ggcttattcg gcaagggtag agatcctgat gtagagggcc
     6901 aaagatacag agaagctgga tggaacgctt acttgaagaa gacaagccaa gtgtaaaata
     6961 tgtaaaattt tgtaaaaatc gtaaaactgt aatgtacatc gttaagtatt tacaaacaac
     7021 tatatgatta tttacaagca gaggcctatt ccaaaatttc gaattccagt aagtcgtcag
     7081 tcaatctggt tctcattatt tcaaaaacca agaggggcac ggatgtagcc ggtattgttg
     7141 tcaatgcatt gcggacaaaa cctttgtatg ccatttgata ccaagtcaac tttgattttg
     7201 tcttcatcct aaggatttgg ttatatgtat caatgtatga gttgtagtac aaggttatgg
     7261 agggtttgta tgaacgcgta gctgatgcat tgtagaaatc aagagcttca agtctagaaa
     7321 gatgaatctt ctgaatcttg gtgattgggt attgtattgc caacaacgag aaagcggcag
     7381 atgcaccagc tacgagaata aaggttgacc tcaatatctt tttggttctc gtacgctcca
     7441 agctcaatag tcgttcgtct atctgttctt cgttcatatt tgagtattgt cgtaacttgc
     7501 ttttgatgga ttctttgctt cgtctataaa cattgacaac cttgaaagtt gctgtatagc
     7561 cttgagtctt gacgaactca aaggtagaga agtagaaggc aaatccaagc gattctttta
     7621 caagcgaaaa actataccca gcaaaaactc caaccaatcc gacttcttta agtttgttca
     7681 aaccgtacat ccagaggttt tggtgtgaac cactcaacat ttctgctgct gtcagtctag
     7741 tatatatggc gtctacaggt gctgctgcga gtgactgaac ggcaccagca ataaaaccag
     7801 ctctccatgt atcgaatggg gagtaataga acaattctcg gggtttatct gcttttgggg
     7861 tatgcagact gttgaatcga tccaaagccg tcaagtaagt tccgtacaat atcaaccctg
     7921 tgagctccat cggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     7981 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     8041 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     8101 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     8161 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     8221 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     8281 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     8341 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     8401 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     8461 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     8521 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     8581 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     8641 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     8701 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     8761 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     8821 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     8881 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     8941 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     9001 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     9061 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     9121 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     9181 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     9241 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     9301 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     9361 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     9421 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     9481 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     9541 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     9601 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     9661 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     9721 cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
