LOCUS       Exported                9369 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses RanCas13b from a SUMO-tagged bacterial expression 
            construct.
ACCESSION   .
VERSION     .
KEYWORDS    pC0062	RanCas13 (B06) His6-TwinStrep-SUMO-BsaI
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9369)
  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 9369)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9369
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             157..240
                     /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             241..531
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG"
     primer_bind     complement(3870..3888)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3884..3931
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(4106..4134)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4161..4179)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4247..4351
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4352..5212
                     /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(4570..4589)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5383..5971
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5872..5891
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6125..6142
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6157..6296
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6222..6241)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6382..6404
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6398..6589)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7901..8983)
                     /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     8945..8964
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8984..9061)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     9267..9286
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg gctagctgga gccatccgca gtttgaaaaa
      181 ggtggtggta gcggtggtgg ttcaggtggt agtgcatggt cacaccctca gtttgagaaa
      241 atgtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct
      301 gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa
      361 aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg
      421 gactccttaa gattcttgta cgacggtatt agaatccaag ctgatcagac ccctgaagat
      481 ttggacatgg aggataacga tattattgag gctcacagag aacagattgg tggatctatg
      541 gagaagcccc tgctgcctaa cgtgtatacc ctgaagcaca agttcttttg gggcgccttc
      601 ctgaatatcg cccggcacaa cgcctttatc accatctgcc acatcaatga gcagctgggc
      661 ctgaaaaccc ccagcaacga cgataagatc gtggacgtgg tgtgcgagac atggaacaat
      721 atcctgaaca atgaccacga tctgctgaag aagtcccagc tgaccgagct gatcctgaag
      781 cacttccctt ttctgacagc catgtgctac caccccccta agaaggaggg caagaagaag
      841 ggccaccaga aggagcagca gaaggagaag gagagcgagg cacagtccca ggcagaggcc
      901 ctgaacccat ccaagctgat cgaggccctg gagatcctgg tgaatcagct gcactctctg
      961 cggaactact atagccacta taagcacaag aagcccgacg ccgagaagga tatcttcaag
     1021 cacctgtata aggccttcga cgcctctctg agaatggtga aggaggatta taaggcccac
     1081 ttcaccgtga atctgacaag ggactttgcc cacctgaacc gcaagggcaa gaacaagcag
     1141 gacaatcccg acttcaacag atacagattc gagaaggatg gcttctttac cgagtctggc
     1201 ctgctgttct ttacaaatct gtttctggac aagagggatg cctattggat gctgaagaag
     1261 gtgtccggct tcaaggcctc tcacaagcag cgcgagaaga tgaccacaga ggtgttttgc
     1321 aggagccgca tcctgctgcc caagctgagg ctggagtccc gctacgacca caaccagatg
     1381 ctgctggata tgctgtctga gctgagcaga tgtcctaagc tgctgtatga gaagctgagc
     1441 gaggagaata agaagcactt ccaggtggag gccgacggct ttctggatga gatcgaggag
     1501 gagcagaacc cattcaagga caccctgatc cggcaccagg atagattccc ctactttgcc
     1561 ctgaggtatc tggacctgaa tgagtccttc aagtctatcc gctttcaggt ggatctgggc
     1621 acataccact attgtatcta cgacaagaag atcggcgatg agcaggagaa gaggcacctg
     1681 acccgcacac tgctgtcctt cggccggctg caggacttta ccgagatcaa cagaccccag
     1741 gagtggaagg ccctgaccaa ggacctggat tacaaggaga catccaatca gcctttcatc
     1801 tctaagacca caccacacta tcacatcacc gacaacaaga tcggctttcg gctgggcaca
     1861 agcaaggagc tgtacccctc cctggagatc aaggatggcg ccaatagaat cgccaagtac
     1921 ccttataact ccggcttcgt ggcccacgcc tttatctctg tgcacgagct gctgcctctg
     1981 atgttctacc agcacctgac cggcaagagc gaggacctgc tgaaggagac agtgcggcac
     2041 atccagagaa tctataagga cttcgaggag gagcggatca ataccatcga ggatctggag
     2101 aaggcaaacc agggcagact gccactggga gcctttccca agcagatgct gggcctgctg
     2161 cagaataagc agcctgatct gtccgagaag gccaagatca agatcgagaa gctgatcgcc
     2221 gagacaaagc tgctgtctca caggctgaac acaaagctga agagctcccc aaagctgggc
     2281 aagcggagag agaagctgat caagacaggc gtgctggccg actggctggt gaaggatttc
     2341 atgcgctttc agcccgtggc ctacgacgcc cagaatcagc ctatcaagtc tagcaaggcc
     2401 aactccaccg agttctggtt tatcaggcgc gccctggccc tgtacggagg agagaagaat
     2461 aggctggagg gctatttcaa gcagaccaac ctgatcggca acacaaatcc acaccccttc
     2521 ctgaacaagt ttaattggaa ggcctgcagg aatctggtgg atttctacca gcagtatctg
     2581 gagcagcgcg agaagtttct ggaggccatc aagaaccagc catgggagcc ctaccagtat
     2641 tgcctgctgc tgaagatccc taaggagaac aggaagaatc tggtgaaggg atgggagcag
     2701 ggaggaatct ctctgccacg gggcctgttt accgaggcca tcagagagac actgtctgag
     2761 gacctgatgc tgagcaagcc aatccgcaag gagatcaaga agcacggccg ggtgggcttc
     2821 atcagcagag ccatcaccct gtactttaag gagaagtatc aggataagca ccagagcttc
     2881 tacaatctgt cctataagct ggaggcaaag gcaccactgc tgaagcggga ggagcactac
     2941 gagtattggc agcagaacaa gcctcagtct ccaaccgaga gccagaggct ggagctgcac
     3001 acaagcgacc gctggaagga ttacctgctg tataagagat ggcagcacct ggagaagaag
     3061 ctgcggctgt accggaatca ggacgtgatg ctgtggctga tgaccctgga gctgacaaag
     3121 aaccacttca aggagctgaa cctgaattat caccagctga agctggagaa tctggccgtg
     3181 aacgtgcagg aggccgatgc caagctgaac cctctgaatc agaccctgcc catggtgctg
     3241 cctgtgaagg tgtaccctgc cacagccttt ggcgaggtgc agtaccacaa gaccccaatc
     3301 aggacagtgt atatccgcga ggagcacacc aaggccctga agatgggcaa cttcaaggcc
     3361 ctggtgaagg accggagact gaatggcctg ttctctttta tcaaggagga gaacgataca
     3421 cagaagcacc caatcagcca gctgcggctg aggcgcgagc tggagatcta ccagtctctg
     3481 agagtggacg ccttcaagga gacactgagc ctggaggaga agctgctgaa taagcacaca
     3541 tccctgtcct ctctggagaa cgagtttagg gccctgctgg aggagtggaa gaaggagtat
     3601 gccgccagct ccatggtgac agacgagcac atcgccttca tcgccagcgt gcgcaatgcc
     3661 ttctgccaca accagtaccc cttctacaag gaggccctgc acgcccctat cccactgttc
     3721 accgtggccc agcccaccac agaggagaag gatggcctgg gaatcgcaga ggccctgctg
     3781 aaggtgctga gggagtactg tgagatcgtg aagtcccaga tctaaggccg cactcgaggc
     3841 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     3901 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggatatcc
     3961 cgcaagaggc ccggcagtac cggcataacc aagcctatgc ctacagcatc cagggtgacg
     4021 gtgccgagga tgacgatgag cgcattgtta gatttcatac acggtgcctg actgcgttag
     4081 caatttaact gtgataaact accgcattaa agcttatcga tgataagctg tcaaacatga
     4141 gaattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     4201 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4261 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4321 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     4381 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     4441 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     4501 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     4561 taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg
     4621 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     4681 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     4741 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     4801 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     4861 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
     4921 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     4981 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     5041 tgataaatct ggagccggtg agcgtgggtc acgcggtatc attgcagcac tggggccaga
     5101 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     5161 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     5221 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5281 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     5341 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5401 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     5461 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5521 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5581 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5641 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     5701 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5761 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5821 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5881 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5941 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     6001 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     6061 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     6121 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     6181 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     6241 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     6301 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     6361 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     6421 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     6481 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     6541 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     6601 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     6661 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     6721 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     6781 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     6841 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     6901 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     6961 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     7021 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga
     7081 tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg
     7141 tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc
     7201 cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg
     7261 acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt
     7321 ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccaa tgatcgaagt
     7381 taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg
     7441 cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat
     7501 aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc
     7561 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     7621 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     7681 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     7741 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     7801 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     7861 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     7921 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     7981 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     8041 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     8101 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     8161 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     8221 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     8281 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     8341 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     8401 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     8461 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     8521 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     8581 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     8641 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     8701 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     8761 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     8821 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     8881 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     8941 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     9001 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     9061 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     9121 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     9181 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     9241 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     9301 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     9361 ccgcgaaat
//
