LOCUS       sequence-413710-       10139 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  empty BLINCAR plasmid for payload delivery, no payload, integrates 
            at CRF1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10139)
  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 10139)
  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: pWR91
FEATURES             Location/Qualifiers
     source          1..10139
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(89..105)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1443..2252
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             3448..5076
                     /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             4398..4406
                     /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)"
     primer_bind     complement(5527..5543)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      complement(8384..8972)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8464..8483)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9143..9934,9935..10003))
                     /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     9766..9785
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10004..10108)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccaatgatac cggtgcgatc gtgggcccta gtacttccct cgaccatgca ttagatcttg
       61 catgcctcta gaccatgatt acggattcac tggccgtcgt tttacaacgt cgtgactggg
      121 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
      181 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
      241 aatggcgctt tgcctggttt ccggcaccag aagcggtgcc ggaaagctgg ctggagtgcg
      301 atcttcctga ggccgatact gtcgtcgtcc cctcaaactg gcagatgcac ggttacgatg
      361 cgcccatcta caccaacgtg acctatccca ttacggtcaa tccgccgttt gttcccacgg
      421 agaatccgac gggttgttac tcgctcacac ttaatgttga tgaaagctgg ctacaggaag
      481 gccagacgcg aattattttt gatggcgtta actcggcgtt tcatctgtgg tgcaacgggc
      541 gctgggtcag ttaaggccag gacagtcgtt tgccgtctga atttgacctg agcgcatttt
      601 tacgcgccgg agaaaaccgc ctcgcggtga tggtgctgcg ctggagtgac ggcagttatc
      661 tggaagatca ggatatgtgg cggataaagt tctacttagt ccccggggat ccgtacactt
      721 attgttaact atgaaacata tacttatgga caaggttcac tgatttggac tgaaaagcaa
      781 aaacgagaaa tgagtcaacg tctgagatca atccctagac cacccgggaa ggtcatgtgt
      841 ttcatgaagt acgataaggt tggtaaccga ttgactcatt ggttcgtggc ggagaagtac
      901 gcagagtaaa accggggccg attcgtggta aattctggaa tgatccagag gcgcgacatt
      961 tatgcagaca atttgtgttt tgtcgcaaac gatgttatag cgaaattttt cactctgtca
     1021 gataaatgga ttttgtcaaa agggggaagt agaaggagaa tgggcccgag atgttctgcc
     1081 aaattctcag tagcataatg tgaaagaagc ccttacattg tccagcctct ggcatcatta
     1141 aaaaccgtag cggaaaccaa ttgtctctgt tcttccctgg cacaccctgg tagccccacc
     1201 cagttgtagt acatctcaca cgctggcaac ttgggacaat cagcaacttt tttttctttt
     1261 aattttttca gcgcgacatt ttgcctcttc tgcgagaaca gactttttca cctccatctc
     1321 accccccttt gcacttatat aaattggacc agttcctccc attgtagaaa aaattttgct
     1381 ggaccttttt ctcttttttt tgtcctttag tttcatacaa tctaagtcta tctacatgta
     1441 caatgggtaa ggaaaagact cacgtttcga ggccgagatt aaattccaac atggatgctg
     1501 atttatatgg gtataaatgg gctagagata atgtcgggca atcaggtgcg acaatctata
     1561 gattgtatgg gaagcccgat gcgccagagt tgtttttgaa acatggcaaa ggtagcgttg
     1621 ccaatgatgt tacagatgag atggtcagac taaactggtt gacggaattt atgcctcttc
     1681 cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc
     1741 ccggcaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg
     1801 atgcgttggc agtgttcttg agaagattgc attcgattcc tgtttgtaat tgtcctttta
     1861 acagcgatag agtatttcgt ctcgctcagg cgcaatcaag aatgaataac ggtttggttg
     1921 atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa
     1981 tgcataagtt gttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg
     2041 ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga agagtcggaa
     2101 tcgcagacag ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt
     2161 cattacagaa aagacttttt caaaaatatg gtattgataa tcctgatatg aataaattgc
     2221 agtttcattt gatgctcgat gagtttttct aagcggccgc taaccacttg caaaatcctt
     2281 tgtattcttg ttctgcaaac attttgtcca atctcttatc tttctacgat gttgaagaac
     2341 atacattttt gttagtccag cttgttatcc tttttttatt aaataaaaaa ttttattgtt
     2401 aaagttgttt ttcttctttg tctgtcctgt tttaactcat atatattcta tatattacag
     2461 aaagaaagta gatcatctaa aataaacatt tatttgatga gacaaagtag gttttttaga
     2521 atacaatgta gcatcctcta atatacaata gagttgtttt ataaaagcga tacatttaat
     2581 attatgacag gactggtgaa gtgtatagaa ttgtgctaat ctggcaaacg aacccaagaa
     2641 gacgttaaag caaatagtgc attttgtatt acaatcaagt tcgtcgagat cctcgttagt
     2701 atttccgtga aggcagccat tgtgaaaatt gaacatcacc tgaggtattt tagccaccag
     2761 aagcggcggt acggaagaaa gtgtgtacga tggttggtgg tggaattgcg tgcatgcctg
     2821 atggggcaat attaattaga tagagctttg gtgatattag tggataatag aattcacaga
     2881 gaagacatca ggagcaattt ccaagagcca ttgatgatgt aattgcccca acagcaagat
     2941 tcagatctga caattgacca ccgttttgta gaagcaaaaa atcgtagatt atcaccaaga
     3001 gggtttttca ccgaaccagc aaatagaaac tatgccgtag aactcgccca ggcttttttg
     3061 ctagcacttt ccagcagtag aaccgtccaa ttaagtcaac aggaaccatt gaggtcgagc
     3121 ccaaccacct gaaccccctc acggtcgtgt ccctattatt gatccagagg gtgccagttt
     3181 cggtagccaa tattggttca tgggtttcta tggcccggag tgagtttgca ggttggcccc
     3241 ggcgccgtct gcaggatggg agttatagcg gccaaacttc acatttcgaa atcctgctgc
     3301 agccaatctg aagaattaat ataaattcgt gtcgaatcgc cgtctgtgaa atttcagtac
     3361 ttgattttct tttcttcttc tttttctctt ttgtttcttc agaatcaata cacatttttt
     3421 cttccctata aacaattcat cggtaccatg gtgaagcgtg agaaaaatgt catctatggc
     3481 cctgagcctc tccatccttt ggaggatttg actgccggcg aaatgttgtt tcgtgctctc
     3541 cgcaagcact ctcatttgcc tcaagccttg gtcgatgtgg tcggcgatga atctttgagc
     3601 tacaaggagt tttttgaggc aaccgtcttg ttggctcagt ccctccacaa ttgtggctac
     3661 aagatgaacg acgtcgttag tatctgtgct gaaaacaata cccgtttctt cattccagtc
     3721 atcgccgcat ggtatatcgg tatgatcgtg gctccagtca acgagagcta cattcccgac
     3781 gaattgtgta aagtcatggg tatctctaag ccacagattg tcttcaccac taagaatatt
     3841 ttgaacaaag tcttggaagt ccaaagccgc accaacttta ttaagcgtat catcatcttg
     3901 gacactgtgg agaatattca cggttgcgaa tctttgccta atttcatctc tcgctattca
     3961 gacggcaaca tcgcaaactt taaaccactc cacttcgacc ctgtggaaca agttgcagcc
     4021 attttgtgta gcagcggtac tactggactc ccaaagggag tcatgcagac ccatcaaaac
     4081 atttgcgtgc gtttgatcca tgctctcgat ccacgtgtgg gcactcagtt gattcctggt
     4141 gtcaccgtct tggtctactt gcctttcttc catgctttcg gctttagcat tactttgggt
     4201 tactttatgg tcggtctccg cgtgattatg ttccgccgtt ttgatcagga ggctttcttg
     4261 aaagccatcc aagattatga agtccgcagt gtcatcaacg tgcctagcgt gatcttgttt
     4321 ttgtctaaga gcccactcgt ggacaagtac gacttgtctt cattgcgtga attgtgttgc
     4381 ggtgccgctc cattggctaa ggaggtcgct gaagtggccg ccaaacgctt gaatcttcca
     4441 ggtattcgtt gtggcttcgg cctcaccgaa tctaccagcg ctaacattca ctctctcggt
     4501 gatgagttta agagcggctc tttgggccgt gtcactccac tcatggctgc taagatcgct
     4561 gatcgcgaaa ctggtaaggc tttgggtccg aaccaagtgg gcgagttgtg tatcaaaggc
     4621 cctatggtga gcaagggtta tgtcaataac gttgaagcta ccaaggaggc catcgacgac
     4681 gacggctggt tgcattctgg tgattttgga tattacgacg aagatgagca tttttacgtc
     4741 gtggatcgtt acaaggagtt gatcaaatac aagggtagcc aggttgctcc agctgagttg
     4801 gaggagattt tgttgaaaaa tccatgcatt cgcgatgtcg ctgtggtcgg cattcctgat
     4861 ttggaggccg gcgaattgcc ttctgctttc gttgtcaagc agcctggtaa agaaattacc
     4921 gccaaagaag tgtatgatta cttggctgaa cgtgtgagcc atactaagta cttgcgtggc
     4981 ggcgtgcgtt ttgttgactc catccctcgt aacgtaacag gcaaaattac ccgcaaggag
     5041 ttgttgaaac aattgttgga gaaggccggc ggttagctta attaagcatt ttagacaagt
     5101 gcctatttat tcagattgat tttttatatt gaatatggat gttgattacg acttgtgtta
     5161 cgaagcatga acatgtgatt tgaaaaagtg tttacgaata aaagaacttt ttttgcaacc
     5221 gttgctagtt gaagtgtaga ctctgaatgc gaagggttgc aaatgtgccc agtagaaaaa
     5281 atattccata tttttctgaa cttaattcaa tgaaatacga gtattactac aactgccaat
     5341 atcggaacaa tttaacgaga gcaagctata taaaatgaat gttcatttac acaagctaga
     5401 accaaaactt gaattaaaat ttaacataac taaatgtaac tagataaaag agagacataa
     5461 ccaaacgttc agaaggcaga actagtaaag ttctacttag tccccgggga ccatgattac
     5521 ggattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     5581 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     5641 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgctttg cctggtttcc
     5701 ggcaccagaa gcggtgccgg aaagctggct ggagtgcgat cttcctgagg ccgatactgt
     5761 cgtcgtcccc tcaaactggc agatgcacgg ttacgatgcg cccatctaca ccaacgtgac
     5821 ctatcccatt acggtcaatc cgccgtttgt tcccacggag aatccgacgg gttgttactc
     5881 gctcacattt aatgttgatg aaagctggct acaggaaggc cagacgcgaa ttatttttga
     5941 tggcgttaac tcggcgtttc atctgtggtg caacgggcgc tgggtcagtt aaggccagga
     6001 cagtcgtttg ccgtctgaat ttgacctgag cgcattttta cgcgccggag aaaaccgcct
     6061 cgcggtgatg gtgctgcgct ggagtgacgg cagttatctg gaagatcagg atatgtggcg
     6121 gatcttaagg tggccaactc atatggcgta cgagaattca gttaactcct gcaggaatcg
     6181 attggccggc ctcacgtgga cgcgttatta atcgtttaaa cctacgtaac cgcggcttaa
     6241 ttaaagctag ccagcgcttg agctcattgg gtgccataat cacaccttaa cttatctttt
     6301 cacatctacg cttacttaaa agccaaaggt cagtactatc cattctcact cgtataaccg
     6361 gtgctttaca actttacgta atttcttcac atccactgcc gttttgactt catccataaa
     6421 atggtattaa tcaacggtgt aaagtatgcc tgtgaacggt gcattcgtgg tcacagagtc
     6481 accacctgcg cccacactga ccagccgtta accatgatca aaccaaaagg aagacccgct
     6541 tcacaatgtc agcattgccg cgaccagcgt aaaatgaaaa acctccatac aacttgtact
     6601 tgtggaaaaa aaggaaaatc tcctggaatg catttggcct cttgtccctg ccacaagaac
     6661 tcacattgta cttgttcagc taatcaggca gcaaaaaaag tgggcgtcgc tcctcaaggc
     6721 aacattcatc accaaacagc ctcggaacga gctaaagaga agtcgttgat cacggcagct
     6781 aacgctgatt tgttgaaaag atctgcctct gcgtcttcga actcgaattt gaatagtaac
     6841 tcgtcgaacg gaaatccttc aacttcacct agaagcctga acggcacatc gtcatcattc
     6901 acagcgtcaa actctcttaa caattacgtg atcgaagatg tcattgtccc ctttgaaacc
     6961 agcaacggct tgtttgactt gttttcctcg gctgctccgt cggaaaacaa cacttccaac
     7021 aacagcatca acggctctaa aactaacctc aacgatccct tcaacaccgg aagtaattcc
     7081 aattctgctg gctcattgag cccaagctct aacttcaaca ataacaataa cactacaatg
     7141 aacggtacta atagtatcag tttaagcaac aacaactaca acgacctctc ttacgacacc
     7201 aataacgaca cagcttacga aaaagactca attgatcttc ccacacttgc aactgatata
     7261 aagcgccact acaataatcc cgatatcgat gcccatttat cacctacaga actcgagttg
     7321 gtggatcaca tgtttccctt gtttccctta gtgggaacag ccagttttga cagcgacctg
     7381 agccagccac ttctgtcaat tccacagaac aacatattca cttctcttca tgaaaaaagt
     7441 gctcctatga gcacaacctt caccaacttg actggtacaa atggaaatgg aaactccaaa
     7501 gttaatcccc tcgtaaactc gaacggaagt aacaattcca atgcaaacgt aacgaatact
     7561 aatgcagaca gtaacgtaag tacggatctt tcgcattcgc tgtctatcaa atctctcaac
     7621 agaccacatc agcccaaacc agtaagacct attccccagc ctagtgctac accgcaatcg
     7681 acttcttcta atgttaactc aaccaatagc ggcaataatc attaccaacc tatacggcct
     7741 aaagggcccg aaggtgtctt atctatagcg tcgaattcgt ccagtcgctc ttttgatgta
     7801 aaccataacc acaactcgtc gtttatagta ggaggaatac ccaactctac aaattctgct
     7861 gcgtatcctc cgagtggact ttatggggcc tatgatgtgc acaatgttgc cagtggcttc
     7921 catagcgacg acagtcctca ttatcaccat tttggaggta caggcttggg gtcaacttct
     7981 tcttcaacga atatagcctc gaatcagaat gaaagtgatt ttgccaactc cgaaaactgg
     8041 ttttctgaac tcgcttcaaa taacaataaa agaaccactt cgttgtcaac atcgtcgtcg
     8101 tttgctaaca tatatgagaa tcagaagaac ggcttatctg aagatcggaa ggtttccggt
     8161 acacctgacg tactgaatgg ggacatctac ggcaacagca aacttactag tgcagtccca
     8221 gaagagaacg gtgaagaaca aaactattct tcttctgtac ctcctgctca ggcaccaatg
     8281 gactatgagg tcaatttgcc gatgtgagct ccatcggaaa gaacatgtga gcaaaaggcc
     8341 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     8401 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     8461 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     8521 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     8581 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     8641 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     8701 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     8761 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     8821 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     8881 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     8941 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     9001 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     9061 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     9121 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     9181 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     9241 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     9301 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     9361 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     9421 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     9481 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     9541 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     9601 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     9661 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     9721 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     9781 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     9841 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     9901 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     9961 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
    10021 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
    10081 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
