LOCUS       sequence-399197-        8705 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses codon optimized miniature mCas13 for protein expression in
            E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8705)
  AUTHORS   Mahas A, Wang Q, Marsic T, Mahfouz MM
  TITLE     A Novel Miniature CRISPR-Cas13 System for SARS-CoV-2 Diagnostics.
  JOURNAL   ACS Synth Biol. 2021 Oct 15;10(10):2541-2551. doi: 
            10.1021/acssynbio.1c00181. Epub 2021 Sep 21.
  PUBMED    34546709
REFERENCE   2  (bases 1 to 8705)
  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: 
            "10.1021/acssynbio.1c00181"; journalName: "ACS Synth Biol"; date: 
            "2021-10-15- 15"; volume: "10"; issue: "10"; pages: "2541-2551"
COMMENT     Sequence Label: mCas13 Twin-SUMO
FEATURES             Location/Qualifiers
     source          1..8705
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1328..2410)
                     /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     2372..2391
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2411..2488)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2694..2713
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2797..2816
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2797..2815
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2816..2840
                     /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             2855..2877
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2896..2913
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2923..2940
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(2953..2976,2977..3012,3013..3036)
                     /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             3037..3330
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(6002..6020)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6016..6063
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6238..6243,6244..6260,6261..6266))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(6293..6311)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6379..6483
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6484..6552,6553..7344)
                     /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(6702..6721)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7515..8103
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8004..8023
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8257..8274
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8289..8428
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8354..8373)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8514..8536
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(8530..8705,1..16))
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
       61 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
      121 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
      181 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
      241 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
      301 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
      361 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
      421 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
      481 gtggccagga cccaacgctg cccgagatgc gccgcgtgcg gctgctggag atggcggacg
      541 cgatggatat gttctgccaa gggttggttt gcgcattcac agttctccgc aagaattgat
      601 tggctccaat tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt ccattcaggt
      661 cgaggtggcc cggctccatg caccgcgacg caacgcgggg aggcagacaa ggtatagggc
      721 ggcgcctaca atccatgcca acccgttcca tgtgctcgcc gaggcggcat aaatcgccgt
      781 gacgatcagc ggtccaatga tcgaagttag gctggtaaga gccgcgagcg atccttgaag
      841 ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg cgggcatccc
      901 gatgccgccg gaagcgagaa gaatcataat ggggaaggcc atccagcctc gcgtcgcgaa
      961 cgccagcaag acgtagccca gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc
     1021 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     1081 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     1141 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     1201 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     1261 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     1321 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     1381 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     1441 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     1501 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     1561 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     1621 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     1681 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     1741 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     1801 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     1861 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     1921 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     1981 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     2041 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     2101 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     2161 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     2221 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     2281 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     2341 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     2401 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     2461 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     2521 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     2581 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     2641 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     2701 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     2761 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat
     2821 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
     2881 accatgggca gcagccatca tcatcatcat cacagcagcg gcctggtgcc gcgcggcagc
     2941 catatggcta gctggagcca tccgcagttt gaaaaaggtg gtggtagcgg tggtggttca
     3001 ggtggtagtg catggtcaca ccctcagttt gagaaaatgt cggactcaga agtcaatcaa
     3061 gaagctaagc cagaggtcaa gccagaagtc aagcctgaga ctcacatcaa tttaaaggtg
     3121 tccgatggat cttcagagat cttcttcaag atcaaaaaga ccactccttt aagaaggctg
     3181 atggaagcgt tcgctaaaag acagggtaag gaaatggact ccttaagatt cttgtacgac
     3241 ggtattagaa tccaagctga tcagacccct gaagatttgg acatggagga taacgatatt
     3301 attgaggctc acagagaaca gattggtgga tccatgggaa tagactatag tctaacatca
     3361 gattgctacc gtggtatcaa taaatcttgt ttcgctgtgg cgctgaacat tgcttacgac
     3421 aactgcgatc ataaaggctg ccgtaccctg ctgtcggagg ttttgcgctc taaaggcggt
     3481 atttcggacg aacagattaa gtcccaagtt gtcgatggta tccagaagcg cctgaaggat
     3541 atccgtaact atttcagcca ttattatcat gcagaggatt gcctgcgctt tggtgaccag
     3601 gatgcggtga aagtgtttct ggaggagatt tataagaatg cagagagcaa aacggtcggt
     3661 gcgaccaaag agtctgacta caagggcgtt gtacctccgc tctttgaatt acataatggt
     3721 acatacatga ttaccgcagc gggtgtcatc tttctcgcca gcttcttctg ccatcgtagc
     3781 aacgtttatc gcatgctggg tgccgtcaaa ggcttcaaac acaccggcaa agagcagctg
     3841 tccgatggcc agaaacgtga ctatggcttt acgcgtcgcc tgctcgctta ctatgctctg
     3901 cgtgactcct acagcgttgg cgcagaagat aaaacgcgtt gctttcgtga aattctgtcg
     3961 tatctgtcac gtgttccaca gttggcggtg gactggctga acgaacaaca attgttgacg
     4021 ccggaagaaa aggaggcgtt tctgaaccag ccggcagagg acgagggcgg tgacatctct
     4081 gacagcagta gctccgacaa aaacaaaaag tccaaagaga aaagacgcag cctgcgccgt
     4141 gatgagaagt tcattttgtt tgccatccaa tttatcgagg gctgggcagc agagcagggt
     4201 ctggatgtta ccttcgctcg ttaccagaag accgttgaaa aggctgagaa caaaaaccag
     4261 gacggtaaac aagcgcgtgc agttcagctc aagtaccgca accaaggcct gaacccggat
     4321 ttcaacaacg agtggatgta ttacatccag aacgagcacg cgattattca gattaaactg
     4381 aataataaga aagcggttgc agcgcgtatt agcgaaaatg aactgaagta tttggtcctg
     4441 ttgatcttcg aggagaaggg taatgacgcg gtgcagaagt tgaattgcta catctacagc
     4501 atgagccaga agattgaggg tgaatggaaa caccgtccgg aagatgagcg ttggatgccg
     4561 agcttcacca aacgagcgga ccgcaccgtg accccggaag ccgtccaaag ccgtctgagc
     4621 tatatccgca aacagctgca agaaaccatt gagaagattg gtcaagaaga accgcgtaac
     4681 aacaagtggc tgatctacaa gggcaaaaag atctctatga ttttgaagtt catcagtgat
     4741 tcgatccgtg atattcaacg ccgtccgaat gttaaacaat atcacatttt gcgtgacgca
     4801 ctgcaacgtc tcgacttcga tggtttttat aaggaacttc agaattatgt taatgacggc
     4861 cgcattgccg taagcctgta tgaccaaatc aagggcgtga atgacatcag cggtctgtgt
     4921 aaaaaagttt gtgaactgac cttggaaagg ctagcgggtc tggaggctaa aaacggcagc
     4981 gaactgagac gttacatcgg cctggaagcg caggagaagc acccgaagta cggtgaatgg
     5041 aataccctgc aagaaaaagc gaaacgcttt ctggagtctc agttctccat tggtaagaat
     5101 tttttgcgta aaatgtttta cggcgattgt tgtcaaaagc gctgcttcga tgaggagaaa
     5161 ggctacaaca cccaggcgaa ggagcgcaaa agcctgtatt ccatcgttaa ggagaagctg
     5221 aaggatatca agccgatcca tgatgaccgt tggtatctga ttgatcgtaa cccgaaaaac
     5281 tacgataata agcacagccg catcatccgt cagatgtgca acacgtacat ccaagatgtg
     5341 ttatgcatga agatggcgat gtggcactac gagaaattaa tctccgcgac tgagttccgt
     5401 aacaagttgg agtggaactg cattggtcag ggtaacatgg gttatgaacg ttactcactg
     5461 tggtacaaga ccggttgcgg tgtggtgatc caattcacgc cagctgattt cctgcggctg
     5521 gacattattg aaaaaccggc aatgatcgag aacatctgcc agtgtttcgt gctgggcaac
     5581 aaaaagctaa acagcggcgc ggaaaagaaa atcacttggg ataaattcaa caaggacggc
     5641 atagcgaaat accgtaagcg ccaggccgag gcggtgcgtg cgatcttcgc gtttgaagag
     5701 ggtctcaaga tccaagaaga caaatggtca cacgaacgtt acttcccgtt ttgtaatatc
     5761 ttggatgaag ccgtgaagca ggggaaaatc aaagataccg gtaaggacaa agaagcctta
     5821 aatcgtggtc gtaacgactt ttttcatgaa gaatttaaaa gcaccgaaga ccagcaagcg
     5881 atttttcaaa aatacttccc gattgttgaa cggaaggacg acaccaaaaa acgtcgcgat
     5941 aagaaacaga agtaagcggc cgcactcgag gcccgaaagg aagctgagtt ggctgctgcc
     6001 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     6061 ttgctgaaag gaggaactat atccggatat cccgcaagag gcccggcagt accggcataa
     6121 ccaagcctat gcctacagca tccagggtga cggtgccgag gatgacgatg agcgcattgt
     6181 tagatttcat acacggtgcc tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt
     6241 aaagcttatc gatgataagc tgtcaaacat gagaattctt gaagacgaaa gggcctcgtg
     6301 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     6361 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     6421 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     6481 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     6541 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     6601 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     6661 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     6721 tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     6781 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     6841 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     6901 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     6961 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     7021 atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     7081 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     7141 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     7201 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     7261 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     7321 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     7381 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     7441 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     7501 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     7561 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     7621 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     7681 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     7741 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     7801 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     7861 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     7921 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     7981 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     8041 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     8101 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     8161 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     8221 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     8281 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     8341 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     8401 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     8461 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     8521 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     8581 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     8641 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     8701 ggttt
//
