LOCUS       sequence-217011-        8946 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 8946)
  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 8946)
  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..8946
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1020..1211
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1205..1227)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1313..1455
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1368..1387
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1470..1487)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1641..2229)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1721..1740)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2400..3191,3192..3260))
                     /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     3023..3042
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3261..3365)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3433..3451
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     3675..3694
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3778..3797
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3778..3796
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3797..3821
                     /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             3836..3858
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3877..3894
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3904..3921
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(3934..3957,3958..3993,3994..4017)
                     /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             4018..4311
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(7221..7239)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7235..7282
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(7457..7462,7463..7479,7480..7485))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     promoter        7495..7571
                     /gene="lacI"
                     /label=lacI promoter
     CDS             7572..8654
                     /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"
     primer_bind     complement(7591..7610)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 accaaacgtt tcggcgagaa gcaggccatt atcgccggca tggcggccga cgcgctgggc
       61 tacgtcttgc tggcgttcgc gacgcgaggc tggatggcct tccccattat gattcttctc
      121 gcttccggcg gcatcgggat gcccgcgttg caggccatgc tgtccaggca ggtagatgac
      181 gaccatcagg gacagcttca aggatcgctc gcggctctta ccagcctaac ttcgatcact
      241 ggaccgctga tcgtcacggc gatttatgcc gcctcggcga gcacatggaa cgggttggca
      301 tggattgtag gcgccgccct ataccttgtc tgcctccccg cgttgcgtcg cggtgcatgg
      361 agccgggcca cctcgacctg aatggaagcc ggcggcacct cgctaacgga ttcaccactc
      421 caagaattgg agccaatcaa ttcttgcgga gaactgtgaa tgcgcaaacc aacccttggc
      481 agaacatatc catcgcgtcc gccatctcca gcagccgcac gcggcgcatc tcgggcagcg
      541 ttgggtcctg gccacgggtg cgcatgatcg tgctcctgtc gttgaggacc cggctaggct
      601 ggcggggttg ccttactggt tagcagaatg aatcaccgat acgcgagcga acgtgaagcg
      661 actgctgctg caaaacgtct gcgacctgag caacaacatg aatggtcttc ggtttccgtg
      721 tttcgtaaag tctggaaacg cggaagtcag cgccctgcac cattatgttc cggatctgca
      781 tcgcaggatg ctgctggcta ccctgtggaa cacctacatc tgtattaacg aagcgctggc
      841 attgaccctg agtgattttt ctctggtccc gccgcatcca taccgccagt tgtttaccct
      901 cacaacgttc cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg agcatcctct
      961 ctcgtttcat cggtatcatt acccccatga acagaaatcc cccttacacg gaggcatcag
     1021 tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc cagacattaa
     1081 cgcttctgga gaaactcaac gagctggacg cggatgaaca ggcagacatc tgtgaatcgc
     1141 ttcacgacca cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg
     1201 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     1261 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca
     1321 tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca
     1381 gattgtactg agagtgcacc atatatgcgg tgtgaaatac cgcacagatg cgtaaggaga
     1441 aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     1501 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     1561 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     1621 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     1681 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     1741 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     1801 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     1861 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     1921 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     1981 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     2041 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     2101 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     2161 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     2221 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     2281 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     2341 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     2401 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     2461 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     2521 agtgctgcaa tgataccgcg tgacccacgc tcaccggctc cagatttatc agcaataaac
     2581 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     2641 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     2701 gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     2761 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     2821 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     2881 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     2941 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     3001 tcttgcccgg cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     3061 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     3121 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     3181 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     3241 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     3301 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     3361 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     3421 ttaacctata aaaataggcg tatcacgagg ccctttcgtc ttcaagaacc gggatctcga
     3481 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     3541 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     3601 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     3661 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     3721 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa
     3781 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt
     3841 ttaactttaa gaaggagata taccatgggc agcagccatc atcatcatca tcacagcagc
     3901 ggcctggtgc cgcgcggcag ccatatggct agctggagcc atccgcagtt tgaaaaaggt
     3961 ggtggtagcg gtggtggttc aggtggtagt gcatggtcac accctcagtt tgagaaaatg
     4021 tcggactcag aagtcaatca agaagctaag ccagaggtca agccagaagt caagcctgag
     4081 actcacatca atttaaaggt gtccgatgga tcttcagaga tcttcttcaa gatcaaaaag
     4141 accactcctt taagaaggct gatggaagcg ttcgctaaaa gacagggtaa ggaaatggac
     4201 tccttaagat tcttgtacga cggtattaga atccaagctg atcagacccc tgaagatttg
     4261 gacatggagg ataacgatat tattgaggct cacagagaac agattggtgg atctatgtcc
     4321 aatgagatcg gcgccttcag agagcaccag tttgcctacg ccccaggaaa cgagaagcag
     4381 gaggaggcca ccttcgccac atacttcaac ctggccctgt ctaatgtgga gggcatgatg
     4441 ttcggagagg tggagtctaa cccagacaag atcgagaaga gcctggatac cctgccccct
     4501 gccatcctga gacagatcgc ctcctttatc tggctgtcta aggaggacca ccccgataag
     4561 gcctactcca ccgaggaggt gaaagtgatc gtgacagacc tggtgcggag actgtgcttc
     4621 tacaggaatt atttcagcca ctgtttctat ctggatacac agtactttta ttccgacgag
     4681 ctggtggata ccacagccat cggcgagaag ctgccttaca atttccacca ctttatcacc
     4741 aacagactgt tcaggtatag cctgccagag atcacactgt ttcgctggaa cgagggcgag
     4801 cggaagtacg agatcctgcg ggacggcctg atcttctttt gctgtctgtt cctgaagagg
     4861 ggacaggcag agcggtttct gaatgagctg agattcttta agaggaccga cgaggagggc
     4921 cgcatcaagc ggaccatctt cacaaagtat tgcacaagag agtctcacaa gcacatcggc
     4981 atcgaggagc aggatttcct gatctttcag gacatcatcg gcgatctgaa tagggtgcct
     5041 aaggtgtgcg acggcgtggt ggatctgagc aaggagaacg agagatacat caagaacagg
     5101 gagacaagca atgagtctga tgagaacaag gcccggtata gactgctgat cagggagaag
     5161 gacaagtttc cctactatct gatgcgctac atcgtggatt tcggcgtgct gccttgcatc
     5221 acctttaagc agaacgacta cagcacaaag gagggccggg gccagttcca ctatcaggat
     5281 gcagcagtgg cacaggagga gaggtgttac aattttgtgg tgcgcaacgg caacgtgtac
     5341 tattcctata tgcctcaggc ccagaacgtg gtgcggatca gcgagctgca gggcaccatc
     5401 tccgtggagg agctgagaaa tatggtgtac gcctctatca atggcaagga cgtgaacaag
     5461 agcgtggagc agtacctgta tcacctgcac ctgctgtatg agaagatcct gaccatctcc
     5521 ggccagacaa tcaaggaggg ccgggtggac gtggaggatt acagacctct gctggataag
     5581 ctgctgctgc gcccagcctc taacggcgag gagctgaggc gcgagctgag gaagctgctg
     5641 ccaaagcgcg tgtgcgacct gctgagcaat cggttcgatt gttctgaggg cgtgagcgcc
     5701 gtggagaaga ggctgaaggc catcctgctg cgccacgagc agctgctgct gagccagaac
     5761 cccgccctgc acatcgacaa gatcaagtcc gtgatcgatt acctgtatct gttcttttct
     5821 gacgatgaga agttccggca gcagcctacc gagaaggccc acagaggcct gaaggacgag
     5881 gagtttcaga tgtaccacta tctggtgggc gactacgatt ctcacccact ggccctgtgg
     5941 aaggagctgg aggccagcgg ccggctgaag ccagagatga gaaagctgac cagcgccaca
     6001 tccctgcacg gcctgtatat gctgtgcctg aagggcaccg tggagtggtg tcggaagcag
     6061 ctgatgtcca tcggcaaggg cacagccaag gtggaggcca tcgccgacag agtgggcctg
     6121 aagctgtacg ataagctgaa ggagtatacc cctgagcagc tggagaggga ggtgaagctg
     6181 gtggtcatgc acggatacgc tgccgccgcc accccaaagc caaaggcaca ggcagcaatc
     6241 ccatctaagc tgacagagct gcggttctat tcctttctgg gcaagagaga gatgtctttc
     6301 gccgccttta tccggcagga caagaaggcc cagaagctgt ggctgagaaa tttctatacc
     6361 gtggagaaca tcaagacact gcagaagagg caggctgccg ccgacgcagc ctgcaagaag
     6421 ctgtataacc tggtgggaga ggtggagcgg gtgcacacca acgataaggt gctggtgctg
     6481 gtggcccaga ggtatcgcga gcggctgctg aatgtgggca gcaagtgtgc cgtgacactg
     6541 gacaaccccg agaggcagca gaagctggcc gacgtgtacg aggtgcagaa cgcctggctg
     6601 tccatccgct tcgacgatct ggactttacc ctgacacacg tgaacctgtc taatctgagg
     6661 aaggcctaca atctgatccc ccgcaagcac atcctggcct tcaaggagta tctggacaat
     6721 agggtgaagc agaagctgtg cgaggagtgt cgcaacgtgc ggagaaagga ggatctgtgc
     6781 acctgctgtt cccctcgcta ctctaatctg acaagctggc tgaaggagaa ccactccgag
     6841 agctccatcg agagggaggc cgccaccatg atgctgctgg acgtggagcg caagctgctg
     6901 agcttcctgc tggatgagag gcgcaaggcc atcatcgagt acggcaagtt catcccattt
     6961 tccgccctgg tgaaggagtg ccggctggcc gacgcaggcc tgtgcggcat cagaaatgac
     7021 gtgctgcacg ataacgtgat cagctacgcc gatgccatcg gcaagctgag cgcctatttt
     7081 cccaaggagg cctccgaggc cgtggagtac atccggagaa caaaggaggt gcgggagcag
     7141 aggcgcgagg agctgatggc caactctagc cagtaaggcc gcactcgagg cccgaaagga
     7201 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     7261 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatatc ccgcaagagg
     7321 cccggcagta ccggcataac caagcctatg cctacagcat ccagggtgac ggtgccgagg
     7381 atgacgatga gcgcattgtt agatttcata cacggtgcct gactgcgtta gcaatttaac
     7441 tgtgataaac taccgcatta aagcttatcg atgataagct gtcaaacatg agaaacacca
     7501 tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca
     7561 gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt
     7621 atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa
     7681 aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg
     7741 cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt
     7801 cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt
     7861 cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc
     7921 aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg
     7981 aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca
     8041 acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat
     8101 tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc
     8161 gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg
     8221 aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca
     8281 tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca
     8341 ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg
     8401 aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg
     8461 ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc
     8521 agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg
     8581 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     8641 aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccggga
     8701 tctcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc
     8761 atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg
     8821 ccggcagcgc tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgatc
     8881 ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt
     8941 cccgcc
//
