LOCUS       sequence-417322-        8072 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  empty BLINCAR plasmid for payload delivery, no payload, no genomic 
            target.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8072)
  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 8072)
  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: pWR88
FEATURES             Location/Qualifiers
     source          1..8072
                     /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(5506..5522)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      complement(6317..6905)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6397..6416)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7076..7867,7868..7936))
                     /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     7699..7718
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7937..8041)
                     /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 tcgctcacat ttaatgttga tgaaagctgg ctacaggaag
      481 gccagacgcg aattattttt gatggcgtta actcggcgtt tcatctgtgg tgcaacgggc
      541 gctgggtcgg ttacggccag 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 tagccccatc
     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 gtgtgtacaa 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 tattccgtag 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 agaatcaatt 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 acttgtgttt
     5161 gaaaaagtgt ttacgaataa aagaactttt tttgcaaccg ttgctagttg aagtgtagac
     5221 tctgaatgcg aagggttgca aatgtgccca gtagaaaaaa tattccatat ttttctgaac
     5281 ttaattcaat gaaatacgag tattactaca actgccaata tcggaacaat ttaacgagag
     5341 caagctatat aaaatgaatg ttcatttaca caagctagaa ccaaaacttg aattaaaatt
     5401 taacataact aaatgtaact agataaaaga gagacataac caaacgttca gaaggcagaa
     5461 ctagtaaagt tctacttagt ccccggggac catgattacg gattcactgg ccgtcgtttt
     5521 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     5581 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     5641 gcgcagcctg aatggcgaat ggcgctttgc ctggtttccg gcaccagaag cggtgccgga
     5701 aagctggctg gagtgcgatc ttcctgaggc cgatactgtc gtcgtcccct caaactggca
     5761 gatgcacggt tacgatgcgc ccatctacac caacgtgacc tatcccatta cggtcaatcc
     5821 gccgtttgtt cccacggaga atccgacggg ttgttactcg ctcacattta atgttgatga
     5881 aagctggcta caggaaggcc agacgcgaat tatttttgat ggcgttaact cggcgtttca
     5941 tctgtggtgc aacgggcgct gggtcggtta cggccaggac agtcgtttgc cgtctgaatt
     6001 tgacctgagc gcatttttac gcgccggaga aaaccgcctc gcggtgatgg tgctgcgctg
     6061 gagtgacggc agttatctgg aagatcagga tatgtggcgg atcttaaggt ggccaactca
     6121 tatggcgtac gagaattcag ttaactcctg caggaatcga ttggccggcc tcacgtggac
     6181 gcgttattaa tcgtttaaac ctacgtaacc gcggcttaat taaagctagc cagcgcttga
     6241 gctccatcgg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     6301 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     6361 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     6421 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     6481 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     6541 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     6601 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     6661 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     6721 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     6781 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6841 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6901 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6961 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     7021 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     7081 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     7141 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     7201 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     7261 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     7321 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     7381 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     7441 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     7501 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     7561 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     7621 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     7681 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     7741 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     7801 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7861 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7921 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7981 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     8041 gcacatttcc ccgaaaagtg ccacctgacg tc
//
