LOCUS       sequence-416567-       10162 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  empty BLINCAR plasmid for payload delivery, no payload, integrates 
            at GSC2 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10162)
  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 10162)
  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: pWR90
FEATURES             Location/Qualifiers
     source          1..10162
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(2420..3008)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2500..2519)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3179..3970,3971..4039))
                     /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     3802..3821
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4040..4144)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4264..4280)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             5618..6427
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             7623..9251
                     /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             8573..8581
                     /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(9702..9718)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gttaaggcca ggacagtcgt ttgccgtctg aatttgacct gagcgcattt ttacgcgccg
       61 gagaaaaccg cctcgcggtg atggtgctgc gctggagtga cggcagttat ctggaagatc
      121 aggatatgtg gcggatctta aggtggccaa ctcatatggc gtacgagaat tcagttaact
      181 cctgcaggaa tcgattggcc ggcctcacgt ggacgcgtta ttaatcgttt aaacctacgt
      241 aaccgcggct taattaaagc tagccagcgc ttgagctcaa ctcctgaaca gccaaaggca
      301 agaaagaaat caagaaaaca atgaaaatgg aaaaaataca gcgctcaagc cattgaacaa
      361 caggaattgg atcataacaa ccaatcggtc tccgtgggtc agtgataggg cgaaacctat
      421 catactcaca aattgttgtc tcatttgtca atgcagctaa gtttactcct accaataaga
      481 acaacttgat cgataaaata ataaacacat tattgaggtg gaatccagga tgagcgtaat
      541 agaaggacaa aaatcgatct aacggaagct gagtacttag atagaaatat tctcgagaga
      601 gcatctgctc acccatgccg gcaccgattt tggtcgtgaa gttaagaata gatccaaagc
      661 caaggtctct tccctttcca cactgcatgt attcacagtg ctttattcta ccacctctca
      721 ccattgcatc cattccagca taaatatcct cattcaaatg aagacccttt tgtgctttag
      781 agacccctcc ccttgttgtc atgaaaatac cattcaaaaa gtccggatgg ccataatgta
      841 gttttcctcc taggtattgc aaagttcgag caaacaaggt tccaaatgtt tgctcctttc
      901 cagcagctac gtcacctagt attccgatat tttctgagaa tatgtattct cttgttccta
      961 ttatagcaac tggatgtgaa tgatcactat cctttaattc atttgaataa gggtcaattg
     1021 gggcagaatt ttcttcgaat tcagctaaca cacttctgat tctgagacac tcctcaagat
     1081 aattatcttg gttggcatca ataagttgaa tatactctcc tctgcagaag atcaatgagt
     1141 tgttttgatt gtcagatttt ccatctccta agataggatt tcctgatagc tttattctat
     1201 acttcggaac tctttcccca ttttccaaca aagcgcaact cccatcaatc aaactcgaat
     1261 agaaagtaga ttcgccagta tcaatgtcaa tttcctcatc caaataagat atctgaagtt
     1321 caggataagt tcgaagaagg aattccttat tttctctttc ttcaggagaa aaattcttca
     1381 agcgctgcat tgaagcaaca attctgaact ttcgaagagc catgacagat gcttcttcta
     1441 atttcccgtt ctcatctttg tattctttgt cactaaggtt ttcaacatca aatgatagtt
     1501 tgattgccct ggaataattc ataaagcctg aaattgtacg atacaatgtt tgcgacctca
     1561 aggatgccca tattcttgtc ctaaggatat attcaggtgt tgctactttg aatccaaccg
     1621 agtaataagg taaatcatct ttcgtctcat tatcgaaagt aggcgatgat gtctcagtgt
     1681 caaattcttc tgctagcaat ttggtatcgc gcacaaaaca agaccattcc aacggatgta
     1741 acagtttcaa gtactctaac attgtgacgt gagagtactg ctcctcctcc cttattattt
     1801 ctctaagaga tagcgttatc ttctcactat aatgaggaat aagtacactg aaagttggca
     1861 tggagttaat aggatttact tcaggcatcg gagttgataa tgactgggca aagaatgtga
     1921 ttctcctttg ggcttcagac tgctcgtaaa acaaggctga ttttaatgat tgatcctctt
     1981 gagaaacaaa aaaggttggc tcttttagaa cagtatctcc ttcgattcca gatacagaaa
     2041 cctgtttgta taacaacttt tgaacatgct ctaacgacaa aagatgctca cggtacatcg
     2101 agattattat ggaattccat acttgtgaga ctagaaattt tgcctttata gtcctatcag
     2161 ccggagaagc aattattttc gagaaaattc tttttggtaa tctcgaaaag atattccgcc
     2221 atggtgtcca tatggaagca ccaatataga atgaacgaca tacagaatag attgtattcc
     2281 agacaatata ccatagatag gtatcaagga aaataaaacc aaatctgtca agtatgtcaa
     2341 cgagctccat cggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2401 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2461 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2521 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2581 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2641 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2701 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2761 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     2821 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     2881 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     2941 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3001 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3061 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3121 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3181 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3241 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3301 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     3361 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3421 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3481 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3541 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3601 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3661 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     3721 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     3781 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     3841 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     3901 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     3961 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4021 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4081 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4141 cgcgcacatt tccccgaaaa gtgccacctg acgtcccaat gataccggtg cgatcgtggg
     4201 ccctagtact tccctcgacc atgcattaga tcttgcatgc ctctagacca tgattacgga
     4261 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     4321 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     4381 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc
     4441 accagaagcg gtgccggaaa gctggctgga gtgcgatctt cctgaggccg atactgtcgt
     4501 cgtcccctca aactggcaga tgcacggtta cgatgcgccc atctacacca acgtgaccta
     4561 tcccattacg gtcaatccgc cgtttgttcc cacggagaat ccgacgggtt gttactcgct
     4621 cacacttaat gttgatgaaa gctggctaca ggaaggccag acgcgaatta tttttgatgg
     4681 cgttaactcg gcgtttcatc tgtggtgcaa cgggcgctgg gtcagttaag gccaggacag
     4741 tcgtttgccg tctgaatttg acctgagcgc atttttacgc gccggagaaa accgcctcgc
     4801 ggtgatggtg ctgcgctgga gtgacggcag ttatctggaa gatcaggata tgtggcggat
     4861 aaagttctac ttagtccccg gggatccgta cacttattgt taactatgaa acatatactt
     4921 atggacaagg ttcactgatt tggactgaaa agcaaaaacg agaaatgagt caacgtctga
     4981 gatcaatccc tagaccaccc gggaaggtca tgtgtttcat gaagtacgat aaggttggta
     5041 accgattgac tcattggttc gtggcggaga agtacgcaga gtaaaaccgg ggccgattcg
     5101 tggtaaattc tggaatgatc cagaggcgcg acatttatgc agacaatttg tgttttgtcg
     5161 caaacgatgt tatagcgaaa tttttcactc tgtcagataa atggattttg tcaaaagggg
     5221 gaagtagaag gagaatgggc ccgagatgtt ctgccaaatt ctcagtagca taatgtgaaa
     5281 gaagccctta cattgtccag cctctggcat cattaaaaac cgtagcggaa accaattgtc
     5341 tctgttcttc cctggcacac cctggtagcc ccacccagtt gtagtacatc tcacacgctg
     5401 gcaacttggg acaatcagca actttttttt cttttaattt tttcagcgcg acattttgcc
     5461 tcttctgcga gaacagactt tttcacctcc atctcacccc cctttgcact tatataaatt
     5521 ggaccagttc ctcccattgt agaaaaaatt ttgctggacc tttttctctt ttttttgtcc
     5581 tttagtttca tacaatctaa gtctatctac atgtacaatg ggtaaggaaa agactcacgt
     5641 ttcgaggccg agattaaatt ccaacatgga tgctgattta tatgggtata aatgggctag
     5701 agataatgtc gggcaatcag gtgcgacaat ctatagattg tatgggaagc ccgatgcgcc
     5761 agagttgttt ttgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt
     5821 cagactaaac tggttgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac
     5881 tcctgatgat gcatggttac tcaccactgc gatccccggc aaaacagcat tccaggtatt
     5941 agaagaatat cctgattcag gtgaaaatat tgttgatgcg ttggcagtgt tcttgagaag
     6001 attgcattcg attcctgttt gtaattgtcc ttttaacagc gatagagtat ttcgtctcgc
     6061 tcaggcgcaa tcaagaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg
     6121 taatggctgg cctgttgaac aagtctggaa agaaatgcat aagttgttgc cattctcacc
     6181 ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa
     6241 attaataggt tgtattgatg ttggaagagt cggaatcgca gacagatacc aggatcttgc
     6301 catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaaagac tttttcaaaa
     6361 atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt
     6421 tttctaagcg gccgctaacc acttgcaaaa tcctttgtat tcttgttctg caaacatttt
     6481 gtccaatctc ttatctttct acgatgttga agaacataca tttttgttag tccagcttgt
     6541 tatccttttt ttattaaata aaaaatttta ttgttaaagt tgtttttctt ctttgtctgt
     6601 cctgttttaa ctcatatata ttctatatat tacagaaaga aagtagatca tctaaaataa
     6661 acatttattt gatgagacaa agtaggtttt ttagaataca atgtagcatc ctctaatata
     6721 caatagagtt gttttataaa agcgatacat ttaatattat gacaggactg gtgaagtgta
     6781 tagaattgtg ctaatctggc aaacgaaccc aagaagacgt taaagcaaat agtgcatttt
     6841 gtattacaat caagttcgtc gagatcctcg ttagtatttc cgtgaaggca gccattgtga
     6901 aaattgaaca tcacctgagg tattttagcc accagaagcg gcggtacgga agaaagtgtg
     6961 tacgatggtt ggtggtggaa ttgcgtgcat gcctgatggg gcaatattaa ttagatagag
     7021 ctttggtgat attagtggat aatagaattc acagagaaga catcaggagc aatttccaag
     7081 agccattgat gatgtaattg ccccaacagc aagattcaga tctgacaatt gaccaccgtt
     7141 ttgtagaagc aaaaaatcgt agattatcac caagagggtt tttcaccgaa ccagcaaata
     7201 gaaactatgc cgtagaactc gcccaggctt ttttgctagc actttccagc agtagaaccg
     7261 tccaattaag tcaacaggaa ccattgaggt cgagcccaac cacctgaacc ccctcacggt
     7321 cgtgtcccta ttattgatcc agagggtgcc agtttcggta gccaatattg gttcatgggt
     7381 ttctatggcc cggagtgagt ttgcaggttg gccccggcgc cgtctgcagg atgggagtta
     7441 tagcggccaa acttcacatt tcgaaatcct gctgcagcca atctgaagaa ttaatataaa
     7501 ttcgtgtcga atcgccgtct gtgaaatttc agtacttgat tttcttttct tcttcttttt
     7561 ctcttttgtt tcttcagaat caatacacat tttttcttcc ctataaacaa ttcatcggta
     7621 ccatggtgaa gcgtgagaaa aatgtcatct atggccctga gcctctccat cctttggagg
     7681 atttgactgc cggcgaaatg ttgtttcgtg ctctccgcaa gcactctcat ttgcctcaag
     7741 ccttggtcga tgtggtcggc gatgaatctt tgagctacaa ggagtttttt gaggcaaccg
     7801 tcttgttggc tcagtccctc cacaattgtg gctacaagat gaacgacgtc gttagtatct
     7861 gtgctgaaaa caatacccgt ttcttcattc cagtcatcgc cgcatggtat atcggtatga
     7921 tcgtggctcc agtcaacgag agctacattc ccgacgaatt gtgtaaagtc atgggtatct
     7981 ctaagccaca gattgtcttc accactaaga atattttgaa caaagtcttg gaagtccaaa
     8041 gccgcaccaa ctttattaag cgtatcatca tcttggacac tgtggagaat attcacggtt
     8101 gcgaatcttt gcctaatttc atctctcgct attcagacgg caacatcgca aactttaaac
     8161 cactccactt cgaccctgtg gaacaagttg cagccatttt gtgtagcagc ggtactactg
     8221 gactcccaaa gggagtcatg cagacccatc aaaacatttg cgtgcgtttg atccatgctc
     8281 tcgatccacg tgtgggcact cagttgattc ctggtgtcac cgtcttggtc tacttgcctt
     8341 tcttccatgc tttcggcttt agcattactt tgggttactt tatggtcggt ctccgcgtga
     8401 ttatgttccg ccgttttgat caggaggctt tcttgaaagc catccaagat tatgaagtcc
     8461 gcagtgtcat caacgtgcct agcgtgatct tgtttttgtc taagagccca ctcgtggaca
     8521 agtacgactt gtcttcattg cgtgaattgt gttgcggtgc cgctccattg gctaaggagg
     8581 tcgctgaagt ggccgccaaa cgcttgaatc ttccaggtat tcgttgtggc ttcggcctca
     8641 ccgaatctac cagcgctaac attcactctc tcggtgatga gtttaagagc ggctctttgg
     8701 gccgtgtcac tccactcatg gctgctaaga tcgctgatcg cgaaactggt aaggctttgg
     8761 gtccgaacca agtgggcgag ttgtgtatca aaggccctat ggtgagcaag ggttatgtca
     8821 ataacgttga agctaccaag gaggccatcg acgacgacgg ctggttgcat tctggtgatt
     8881 ttggatatta cgacgaagat gagcattttt acgtcgtgga tcgttacaag gagttgatca
     8941 aatacaaggg tagccaggtt gctccagctg agttggagga gattttgttg aaaaatccat
     9001 gcattcgcga tgtcgctgtg gtcggcattc ctgatttgga ggccggcgaa ttgccttctg
     9061 ctttcgttgt caagcagcct ggtaaagaaa ttaccgccaa agaagtgtat gattacttgg
     9121 ctgaacgtgt gagccatact aagtacttgc gtggcggcgt gcgttttgtt gactccatcc
     9181 ctcgtaacgt aacaggcaaa attacccgca aggagttgtt gaaacaattg ttggagaagg
     9241 ccggcggtta gcttaattaa gcattttaga caagtgccta tttattcaga ttgatttttt
     9301 atattgaata tggatgttga ttacgacttg tgttacgaag catgaacatg tgatttgaaa
     9361 aagtgtttac gaataaaaga actttttttg caaccgttgc tagttgaagt gtagactctg
     9421 aatgcgaagg gttgcaaatg tgcccagtag aaaaaatatt ccatattttt ctgaacttaa
     9481 ttcaatgaaa tacgagtatt actacaactg ccaatatcgg aacaatttaa cgagagcaag
     9541 ctatataaaa tgaatgttca tttacacaag ctagaaccaa aacttgaatt aaaatttaac
     9601 ataactaaat gtaactagat aaaagagaga cataaccaaa cgttcagaag gcagaactag
     9661 taaagttcta cttagtcccc ggggaccatg attacggatt cactggccgt cgttttacaa
     9721 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     9781 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     9841 agcctgaatg gcgaatggcg ctttgcctgg tttccggcac cagaagcggt gccggaaagc
     9901 tggctggagt gcgatcttcc tgaggccgat actgtcgtcg tcccctcaaa ctggcagatg
     9961 cacggttacg atgcgcccat ctacaccaac gtgacctatc ccattacggt caatccgccg
    10021 tttgttccca cggagaatcc gacgggttgt tactcgctca catttaatgt tgatgaaagc
    10081 tggctacagg aaggccagac gcgaattatt tttgatggcg ttaactcggc gtttcatctg
    10141 tggtgcaacg ggcgctgggt ca
//
