LOCUS       sequence-224746-        8943 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AspCas13b from a SUMO-tagged bacterial expression 
            construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8943)
  AUTHORS   Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang 
            F
  TITLE     Multiplexed and portable nucleic acid detection platform with Cas13,
            Cas12a, and Csm6.
  JOURNAL   Science. 2018 Apr 27;360(6387):439-444. doi: 
            10.1126/science.aaq0179. Epub 2018 Feb 15.
  PUBMED    29449508
REFERENCE   2  (bases 1 to 8943)
  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"; doi: 
            "10.1126/science.aaq0179"; journalName: "Science"; date: 
            "2018-04-27- 27"; volume: "360"; issue: "6387"; pages: "439-444"
COMMENT     Sequence Label: pC0060	AspCas13 (B04) His6-TwinStrep-SUMO-BsaI
FEATURES             Location/Qualifiers
     source          1..8943
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     937..956
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(976..1053)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1259..1278
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1362..1381
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1362..1380
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1381..1405
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             1420..1442
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1461..1478
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1488..1505
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(1518..1541,1542..1577,1578..1601)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             1602..1895
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(4805..4823)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4819..4866
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5041..5046,5047..5063,5064..5069))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(5096..5114)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5182..5286
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5287..5355,5356..6147)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5505..5524)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6318..6906
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6807..6826
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7060..7077
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7092..7231
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7157..7176)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7317..7339
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7333..7524)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(8836..8943,1..975))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
       61 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
      121 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
      181 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
      241 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
      301 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
      361 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
      421 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
      481 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
      541 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
      601 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
      661 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
      721 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
      781 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
      841 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
      901 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
      961 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     1021 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     1081 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     1141 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     1201 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     1261 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     1321 tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg
     1381 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
     1441 gatataccat gggcagcagc catcatcatc atcatcacag cagcggcctg gtgccgcgcg
     1501 gcagccatat ggctagctgg agccatccgc agtttgaaaa aggtggtggt agcggtggtg
     1561 gttcaggtgg tagtgcatgg tcacaccctc agtttgagaa aatgtcggac tcagaagtca
     1621 atcaagaagc taagccagag gtcaagccag aagtcaagcc tgagactcac atcaatttaa
     1681 aggtgtccga tggatcttca gagatcttct tcaagatcaa aaagaccact cctttaagaa
     1741 ggctgatgga agcgttcgct aaaagacagg gtaaggaaat ggactcctta agattcttgt
     1801 acgacggtat tagaatccaa gctgatcaga cccctgaaga tttggacatg gaggataacg
     1861 atattattga ggctcacaga gaacagattg gtggatctat gtccaatgag atcggcgcct
     1921 tcagagagca ccagtttgcc tacgccccag gaaacgagaa gcaggaggag gccaccttcg
     1981 ccacatactt caacctggcc ctgtctaatg tggagggcat gatgttcgga gaggtggagt
     2041 ctaacccaga caagatcgag aagagcctgg ataccctgcc ccctgccatc ctgagacaga
     2101 tcgcctcctt tatctggctg tctaaggagg accaccccga taaggcctac tccaccgagg
     2161 aggtgaaagt gatcgtgaca gacctggtgc ggagactgtg cttctacagg aattatttca
     2221 gccactgttt ctatctggat acacagtact tttattccga cgagctggtg gataccacag
     2281 ccatcggcga gaagctgcct tacaatttcc accactttat caccaacaga ctgttcaggt
     2341 atagcctgcc agagatcaca ctgtttcgct ggaacgaggg cgagcggaag tacgagatcc
     2401 tgcgggacgg cctgatcttc ttttgctgtc tgttcctgaa gaggggacag gcagagcggt
     2461 ttctgaatga gctgagattc tttaagagga ccgacgagga gggccgcatc aagcggacca
     2521 tcttcacaaa gtattgcaca agagagtctc acaagcacat cggcatcgag gagcaggatt
     2581 tcctgatctt tcaggacatc atcggcgatc tgaatagggt gcctaaggtg tgcgacggcg
     2641 tggtggatct gagcaaggag aacgagagat acatcaagaa cagggagaca agcaatgagt
     2701 ctgatgagaa caaggcccgg tatagactgc tgatcaggga gaaggacaag tttccctact
     2761 atctgatgcg ctacatcgtg gatttcggcg tgctgccttg catcaccttt aagcagaacg
     2821 actacagcac aaaggagggc cggggccagt tccactatca ggatgcagca gtggcacagg
     2881 aggagaggtg ttacaatttt gtggtgcgca acggcaacgt gtactattcc tatatgcctc
     2941 aggcccagaa cgtggtgcgg atcagcgagc tgcagggcac catctccgtg gaggagctga
     3001 gaaatatggt gtacgcctct atcaatggca aggacgtgaa caagagcgtg gagcagtacc
     3061 tgtatcacct gcacctgctg tatgagaaga tcctgaccat ctccggccag acaatcaagg
     3121 agggccgggt ggacgtggag gattacagac ctctgctgga taagctgctg ctgcgcccag
     3181 cctctaacgg cgaggagctg aggcgcgagc tgaggaagct gctgccaaag cgcgtgtgcg
     3241 acctgctgag caatcggttc gattgttctg agggcgtgag cgccgtggag aagaggctga
     3301 aggccatcct gctgcgccac gagcagctgc tgctgagcca gaaccccgcc ctgcacatcg
     3361 acaagatcaa gtccgtgatc gattacctgt atctgttctt ttctgacgat gagaagttcc
     3421 ggcagcagcc taccgagaag gcccacagag gcctgaagga cgaggagttt cagatgtacc
     3481 actatctggt gggcgactac gattctcacc cactggccct gtggaaggag ctggaggcca
     3541 gcggccggct gaagccagag atgagaaagc tgaccagcgc cacatccctg cacggcctgt
     3601 atatgctgtg cctgaagggc accgtggagt ggtgtcggaa gcagctgatg tccatcggca
     3661 agggcacagc caaggtggag gccatcgccg acagagtggg cctgaagctg tacgataagc
     3721 tgaaggagta tacccctgag cagctggaga gggaggtgaa gctggtggtc atgcacggat
     3781 acgctgccgc cgccacccca aagccaaagg cacaggcagc aatcccatct aagctgacag
     3841 agctgcggtt ctattccttt ctgggcaaga gagagatgtc tttcgccgcc tttatccggc
     3901 aggacaagaa ggcccagaag ctgtggctga gaaatttcta taccgtggag aacatcaaga
     3961 cactgcagaa gaggcaggct gccgccgacg cagcctgcaa gaagctgtat aacctggtgg
     4021 gagaggtgga gcgggtgcac accaacgata aggtgctggt gctggtggcc cagaggtatc
     4081 gcgagcggct gctgaatgtg ggcagcaagt gtgccgtgac actggacaac cccgagaggc
     4141 agcagaagct ggccgacgtg tacgaggtgc agaacgcctg gctgtccatc cgcttcgacg
     4201 atctggactt taccctgaca cacgtgaacc tgtctaatct gaggaaggcc tacaatctga
     4261 tcccccgcaa gcacatcctg gccttcaagg agtatctgga caatagggtg aagcagaagc
     4321 tgtgcgagga gtgtcgcaac gtgcggagaa aggaggatct gtgcacctgc tgttcccctc
     4381 gctactctaa tctgacaagc tggctgaagg agaaccactc cgagagctcc atcgagaggg
     4441 aggccgccac catgatgctg ctggacgtgg agcgcaagct gctgagcttc ctgctggatg
     4501 agaggcgcaa ggccatcatc gagtacggca agttcatccc attttccgcc ctggtgaagg
     4561 agtgccggct ggccgacgca ggcctgtgcg gcatcagaaa tgacgtgctg cacgataacg
     4621 tgatcagcta cgccgatgcc atcggcaagc tgagcgccta ttttcccaag gaggcctccg
     4681 aggccgtgga gtacatccgg agaacaaagg aggtgcggga gcagaggcgc gaggagctga
     4741 tggccaactc tagccagtaa ggccgcactc gaggcccgaa aggaagctga gttggctgct
     4801 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     4861 tttttgctga aaggaggaac tatatccgga tatcccgcaa gaggcccggc agtaccggca
     4921 taaccaagcc tatgcctaca gcatccaggg tgacggtgcc gaggatgacg atgagcgcat
     4981 tgttagattt catacacggt gcctgactgc gttagcaatt taactgtgat aaactaccgc
     5041 attaaagctt atcgatgata agctgtcaaa catgagaatt cttgaagacg aaagggcctc
     5101 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     5161 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5221 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     5281 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     5341 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     5401 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     5461 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     5521 ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     5581 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     5641 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     5701 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     5761 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     5821 acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     5881 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     5941 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     6001 gggtcacgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     6061 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     6121 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     6181 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     6241 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     6301 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     6361 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     6421 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     6481 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     6541 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6601 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6661 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     6721 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     6781 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     6841 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     6901 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     6961 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     7021 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     7081 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     7141 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     7201 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     7261 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     7321 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     7381 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     7441 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     7501 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     7561 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     7621 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     7681 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     7741 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     7801 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     7861 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     7921 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     7981 gcgcacccgt ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc tgctggagat
     8041 ggcggacgcg atggatatgt tctgccaagg gttggtttgc gcattcacag ttctccgcaa
     8101 gaattgattg gctccaattc ttggagtggt gaatccgtta gcgaggtgcc gccggcttcc
     8161 attcaggtcg aggtggcccg gctccatgca ccgcgacgca acgcggggag gcagacaagg
     8221 tatagggcgg cgcctacaat ccatgccaac ccgttccatg tgctcgccga ggcggcataa
     8281 atcgccgtga cgatcagcgg tccaatgatc gaagttaggc tggtaagagc cgcgagcgat
     8341 ccttgaagct gtccctgatg gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg
     8401 ggcatcccga tgccgccgga agcgagaaga atcataatgg ggaaggccat ccagcctcgc
     8461 gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg ccatgccggc gataatggcc
     8521 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     8581 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     8641 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     8701 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     8761 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     8821 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     8881 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     8941 tct
//
