LOCUS       sequence-409748-        8118 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BLINCAR plasmid for gene deletion, no genomic target.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8118)
  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 8118)
  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: pWR95
FEATURES             Location/Qualifiers
     source          1..8118
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(114..130)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1468..2277
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             3473..5101
                     /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             4423..4431
                     /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(5552..5568)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      complement(6363..6951)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6443..6462)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7122..7913,7914..7982))
                     /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     7745..7764
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7983..8087)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccaatgatac cggtgcgatc gtgggcccta gtacttccct cgaggaacgt tgtgcccggg
       61 agattcgacc atgcattaga tcttgcatgc ctctagacca tgattacgga ttcactggcc
      121 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
      181 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
      241 caacagttgc gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc accagaagcg
      301 gtgccggaaa gctggctgga gtgcgatctt cctgaggccg atactgtcgt cgtcccctca
      361 aactggcaga tgcacggtta cgatgcgccc atctacacca acgtgaccta tcccattacg
      421 gtcaatccgc cgtttgttcc cacggagaat ccgacgggtt gttactcgct cacacttaat
      481 gttgatgaaa gctggctaca ggaaggccag acgcgaatta tttttgatgg cgttaactcg
      541 gcgtttcatc tgtggtgcaa cgggcgctgg gtcagttaag gccaggacag tcgtttgccg
      601 tctgaatttg acctgagcgc atttttacgc gccggagaaa accgcctcgc ggtgatggtg
      661 ctgcgctgga gtgacggcag ttatctggaa gatcaggata tgtggcggat aaagttctac
      721 ttagtccccg gggatccgta cacttattgt taactatgaa acatatactt atggacaagg
      781 ttcactgatt tggactgaaa agcaaaaacg agaaatgagt caacgtctga gatcaatccc
      841 tagaccaccc gggaaggtca tgtgtttcat gaagtacgat aaggttggta accgattgac
      901 tcattggttc gtggcggaga agtacgcaga gtaaaaccgg ggccgattcg tggtaaattc
      961 tggaatgatc cagaggcgcg acatttatgc agacaatttg tgttttgtcg caaacgatgt
     1021 tatagcgaaa tttttcactc tgtcagataa atggattttg tcaaaagggg gaagtagaag
     1081 gagaatgggc ccgagatgtt ctgccaaatt ctcagtagca taatgtgaaa gaagccctta
     1141 cattgtccag cctctggcat cattaaaaac cgtagcggaa accaattgtc tctgttcttc
     1201 cctggcacac cctggtagcc ccacccagtt gtagtacatc tcacacgctg gcaacttggg
     1261 acaatcagca actttttttt cttttaattt tttcagcgcg acattttgcc tcttctgcga
     1321 gaacagactt tttcacctcc atctcacccc cctttgcact tatataaatt ggaccagttc
     1381 ctcccattgt agaaaaaatt ttgctggacc tttttctctt ttttttgtcc tttagtttca
     1441 tacaatctaa gtctatctac atgtacaatg ggtaaggaaa agactcacgt ttcgaggccg
     1501 agattaaatt ccaacatgga tgctgattta tatgggtata aatgggctag agataatgtc
     1561 gggcaatcag gtgcgacaat ctatagattg tatgggaagc ccgatgcgcc agagttgttt
     1621 ttgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac
     1681 tggttgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat
     1741 gcatggttac tcaccactgc gatccccggc aaaacagcat tccaggtatt agaagaatat
     1801 cctgattcag gtgaaaatat tgttgatgcg ttggcagtgt tcttgagaag attgcattcg
     1861 attcctgttt gtaattgtcc ttttaacagc gatagagtat ttcgtctcgc tcaggcgcaa
     1921 tcaagaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg
     1981 cctgttgaac aagtctggaa agaaatgcat aagttgttgc cattctcacc ggattcagtc
     2041 gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt
     2101 tgtattgatg ttggaagagt cggaatcgca gacagatacc aggatcttgc catcctatgg
     2161 aactgcctcg gtgagttttc tccttcatta cagaaaagac tttttcaaaa atatggtatt
     2221 gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaagcg
     2281 gccgctaacc acttgcaaaa tcctttgtat tcttgttctg caaacatttt gtccaatctc
     2341 ttatctttct acgatgttga agaacataca tttttgttag tccagcttgt tatccttttt
     2401 ttattaaata aaaaatttta ttgttaaagt tgtttttctt ctttgtctgt cctgttttaa
     2461 ctcatatata ttctatatat tacagaaaga aagtagatca tctaaaataa acatttattt
     2521 gatgagacaa agtaggtttt ttagaataca atgtagcatc ctctaatata caatagagtt
     2581 gttttataaa agcgatacat ttaatattat gacaggactg gtgaagtgta tagaattgtg
     2641 ctaatctggc aaacgaaccc aagaagacgt taaagcaaat agtgcatttt gtattacaat
     2701 caagttcgtc gagatcctcg ttagtatttc cgtgaaggca gccattgtga aaattgaaca
     2761 tcacctgagg tattttagcc accagaagcg gcggtacgga agaaagtgtg tacgatggtt
     2821 ggtggtggaa ttgcgtgcat gcctgatggg gcaatattaa ttagatagag ctttggtgat
     2881 attagtggat aatagaattc acagagaaga catcaggagc aatttccaag agccattgat
     2941 gatgtaattg ccccaacagc aagattcaga tctgacaatt gaccaccgtt ttgtagaagc
     3001 aaaaaatcgt agattatcac caagagggtt tttcaccgaa ccagcaaata gaaactatgc
     3061 cgtagaactc gcccaggctt ttttgctagc actttccagc agtagaaccg tccaattaag
     3121 tcaacaggaa ccattgaggt cgagcccaac cacctgaacc ccctcacggt cgtgtcccta
     3181 ttattgatcc agagggtgcc agtttcggta gccaatattg gttcatgggt ttctatggcc
     3241 cggagtgagt ttgcaggttg gccccggcgc cgtctgcagg atgggagtta tagcggccaa
     3301 acttcacatt tcgaaatcct gctgcagcca atctgaagaa ttaatataaa ttcgtgtcga
     3361 atcgccgtct gtgaaatttc agtacttgat tttcttttct tcttcttttt ctcttttgtt
     3421 tcttcagaat caatacacat tttttcttcc ctataaacaa ttcatcggta ccatggtgaa
     3481 gcgtgagaaa aatgtcatct atggccctga gcctctccat cctttggagg atttgactgc
     3541 cggcgaaatg ttgtttcgtg ctctccgcaa gcactctcat ttgcctcaag ccttggtcga
     3601 tgtggtcggc gatgaatctt tgagctacaa ggagtttttt gaggcaaccg tcttgttggc
     3661 tcagtccctc cacaattgtg gctacaagat gaacgacgtc gttagtatct gtgctgaaaa
     3721 caatacccgt ttcttcattc cagtcatcgc cgcatggtat atcggtatga tcgtggctcc
     3781 agtcaacgag agctacattc ccgacgaatt gtgtaaagtc atgggtatct ctaagccaca
     3841 gattgtcttc accactaaga atattttgaa caaagtcttg gaagtccaaa gccgcaccaa
     3901 ctttattaag cgtatcatca tcttggacac tgtggagaat attcacggtt gcgaatcttt
     3961 gcctaatttc atctctcgct attcagacgg caacatcgca aactttaaac cactccactt
     4021 cgaccctgtg gaacaagttg cagccatttt gtgtagcagc ggtactactg gactcccaaa
     4081 gggagtcatg cagacccatc aaaacatttg cgtgcgtttg atccatgctc tcgatccacg
     4141 tgtgggcact cagttgattc ctggtgtcac cgtcttggtc tacttgcctt tcttccatgc
     4201 tttcggcttt agcattactt tgggttactt tatggtcggt ctccgcgtga ttatgttccg
     4261 ccgttttgat caggaggctt tcttgaaagc catccaagat tatgaagtcc gcagtgtcat
     4321 caacgtgcct agcgtgatct tgtttttgtc taagagccca ctcgtggaca agtacgactt
     4381 gtcttcattg cgtgaattgt gttgcggtgc cgctccattg gctaaggagg tcgctgaagt
     4441 ggccgccaaa cgcttgaatc ttccaggtat tcgttgtggc ttcggcctca ccgaatctac
     4501 cagcgctaac attcactctc tcggtgatga gtttaagagc ggctctttgg gccgtgtcac
     4561 tccactcatg gctgctaaga tcgctgatcg cgaaactggt aaggctttgg gtccgaacca
     4621 agtgggcgag ttgtgtatca aaggccctat ggtgagcaag ggttatgtca ataacgttga
     4681 agctaccaag gaggccatcg acgacgacgg ctggttgcat tctggtgatt ttggatatta
     4741 cgacgaagat gagcattttt acgtcgtgga tcgttacaag gagttgatca aatacaaggg
     4801 tagccaggtt gctccagctg agttggagga gattttgttg aaaaatccat gcattcgcga
     4861 tgtcgctgtg gtcggcattc ctgatttgga ggccggcgaa ttgccttctg ctttcgttgt
     4921 caagcagcct ggtaaagaaa ttaccgccaa agaagtgtat gattacttgg ctgaacgtgt
     4981 gagccatact aagtacttgc gtggcggcgt gcgttttgtt gactccatcc ctcgtaacgt
     5041 aacaggcaaa attacccgca aggagttgtt gaaacaattg ttggagaagg ccggcggtta
     5101 gcttaattaa gcattttaga caagtgccta tttattcaga ttgatttttt atattgaata
     5161 tggatgttga ttacgacttg tgttacgaag catgaacatg tgatttgaaa aagtgtttac
     5221 gaataaaaga actttttttg caaccgttgc tagttgaagt gtagactctg aatgcgaagg
     5281 gttgcaaatg tgcccagtag aaaaaatatt ccatattttt ctgaacttaa ttcaatgaaa
     5341 tacgagtatt actacaactg ccaatatcgg aacaatttaa cgagagcaag ctatataaaa
     5401 tgaatgttca tttacacaag ctagaaccaa aacttgaatt aaaatttaac ataactaaat
     5461 gtaactagat aaaagagaga cataaccaaa cgttcagaag gcagaactag taaagttcta
     5521 cttagtcccc ggggaccatg attacggatt cactggccgt cgttttacaa cgtcgtgact
     5581 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     5641 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     5701 gcgaatggcg ctttgcctgg tttccggcac cagaagcggt gccggaaagc tggctggagt
     5761 gcgatcttcc tgaggccgat actgtcgtcg tcccctcaaa ctggcagatg cacggttacg
     5821 atgcgcccat ctacaccaac gtgacctatc ccattacggt caatccgccg tttgttccca
     5881 cggagaatcc gacgggttgt tactcgctca catttaatgt tgatgaaagc tggctacagg
     5941 aaggccagac gcgaattatt tttgatggcg ttaactcggc gtttcatctg tggtgcaacg
     6001 ggcgctgggt cagttaaggc caggacagtc gtttgccgtc tgaatttgac ctgagcgcat
     6061 ttttacgcgc cggagaaaac cgcctcgcgg tgatggtgct gcgctggagt gacggcagtt
     6121 atctggaaga tcaggatatg tggcggatct taaggtggcc aactcatatg gcgtacgaga
     6181 attcagttaa ctcctgcagg aatcgattgg ccggcctcac gtggacgcgt tattaatcgt
     6241 ttaaacctac gtaaccgcgg cttaattaaa gctagccagc gcttgagctc catcggaaag
     6301 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     6361 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     6421 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     6481 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     6541 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     6601 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     6661 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     6721 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     6781 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     6841 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     6901 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     6961 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     7021 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     7081 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     7141 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     7201 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     7261 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     7321 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     7381 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     7441 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     7501 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     7561 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     7621 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     7681 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     7741 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     7801 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     7861 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     7921 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     7981 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     8041 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     8101 aaagtgccac ctgacgtc
//
