LOCUS       sequence-338350-        8601 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of BrCas12b.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8601)
  AUTHORS   Nguyen LT, Macaluso NC, Pizzano BLM, Cash MN, Spacek J, Karasek J, 
            Dinglasan RR, Salemi M, Jain PK
  TITLE     A Thermostable Cas12b from Brevibacillus Leverages One-pot Detection
            of SARS-CoV-2 Variants of Concern
  JOURNAL   medRxiv
REFERENCE   2  (bases 1 to 8601)
  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: "medRxiv"
COMMENT     Sequence Label: PET28a+_BrCas12b
FEATURES             Location/Qualifiers
     source          1..8601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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             148..180
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             3466..3483
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3536..3554)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3550..3597
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3634..4089
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3721..3740)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3931..3952
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4181..4996)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4181..4996)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4904..4923
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4988..5007
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5118..5706
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5607..5626
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5860..5877
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5892..6031
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5957..5976)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6117..6139
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6133..6324)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7133..8215)
                     /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     8177..8196
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8216..8293)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8499..8518
                     /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 gctagcatga ctggtggaca gcaaatgggt
      181 cgcggatcca tgcccgtaag gtcatttaaa gttaagctag tgactcggtc tggcgatgcg
      241 gagcacatgc tgcaactgcg tagggggctg tggaaaaccc atgaaatcgt aaatcagggc
      301 attgcgtact acatgaacaa gttggcgctg atgcgtcaag aaccgtatgc tggtaaaagc
      361 cgtgaggttg ttcgtttgga gctgctgcat agcctgcgtg cacagcagaa acgtaataac
      421 tggaccggcg acgccggtac ggatgatgaa attctgaacc tgagccgtcg cctgtacgag
      481 ttgctcgtgc cgagcgcgat cggcgagaag ggcgatgcgc agatgttatc tcgcaagttt
      541 ctgtcgccgc tggttgatcc gaattctgaa ggtggtaaag gcacggcgaa gtccggccgt
      601 aagccgcgtt ggatgaaaat gcgggaggag ggtcatccgg attgggaagc tgaacgcgag
      661 aaggaccgcg caaagaaagc agcggacccg actgcgtcta ttctgaatga tttggaagcg
      721 tttggtctgc gtccgctgtt cccgctgttc accgacgagc aaaaaggcat ccagtggctg
      781 ccgaaacaaa aacgtcagtt cgtgcgtacc ttcgatcgtg atatgtttca gcaggctctg
      841 gagcgtatgc tgtcctggga gtcctggaac cgccgtgtcg ccgaggaata tcagaagctg
      901 caggctcaac gtgacgagtt gtacgcaaaa tatctggccg atggtggtgc ctggctggaa
      961 gcgttacaaa gctttgagaa gcagcgtgaa gttgaactgg ctgaggagtc ttttgccgct
     1021 aaatccgaat atctgatcac ccgacgacaa attcgcggtt ggaaacaggt gtacgaaaag
     1081 tggtcacaac tgccagagca cgccgcccaa gaacaattct ggcaagttgt agcggacgta
     1141 cagacgtctc tgcctggtgc gtttggtgac ccaaaagttt accagtttct gagccagccg
     1201 gagcatcacc acatctggcg cggttacccg aaccgactat tccactattc tgattacaac
     1261 ggtgttcgca agaagctgca aagagcgcgt cacgatgcga cttttacctt gccggacccg
     1321 gttgaacacc cgctttggat cagattcgat gcgcgtggtg gtaatattca tgattatgag
     1381 atcagccaga atggcaagca atatcaagtt accttctccc gcctcctgtg gcctgaaaat
     1441 gaaacctggg tggagcgtga aaacgttacc gttgcaatcg gggcaagcca gcagcttaag
     1501 cgtcagattc ggttggacgg ctacgccgat aaaaagcaga aggtacgtta tcgtgactac
     1561 tctagcggca tcgagctgac cggtgttctg ggtggtgcga agatccagtt cgaccgtcgt
     1621 cacctgcgca aagcaagtaa tcgtttagcc gacggtgaaa ccggccctgt gtacctgaac
     1681 gtggtggtcg atatcgagcc gtttctggct atgcgcaacg gtcgtctaca aacgccgatt
     1741 ggccaagtgc tgcaagtcaa caccaaggac tggccgaagg tgacgggtta taaaccggct
     1801 gagctgatca gctggattca aaacagcccg ctggcggtgg gtacaggcgt caacaccatt
     1861 gaggcgggta tgcgcgttat gagtgttgat ctgggtcaaa gaagcgcagc tgctgtgagc
     1921 attttcgagg tcatgcgcca gaaaccggcg gaacaggaaa ccaaactctt ctatccgatt
     1981 gctgtgaccg gtctgtatgc ggtgcatcgt cgtagtttgc tgctgcgtct tccaggcgag
     2041 aaaattagcg acgaaatcga acagcaacgt aaaatcagag cccatgctcg tagcttggtg
     2101 cgctaccaga tccgtctgct tgcggacgta ctgcgtctcc acacccgtgg caccgcggaa
     2161 cagagacgtg cgaaactgga tgaattgctg gctaccctgc aaacgaaaca agagttagat
     2221 cagaagttat ggcaaactga gttggaaaag ctgttcgact acattcacga accggcggag
     2281 cgctggcagc aggcgctggt ggcggcgcac cgtaccttgg agccggttat cggtcaggca
     2341 gttcgtcatt ggcgtaagtc cctccgcatt gatcgtaagg gcttggcggg catgagcatg
     2401 tggaacattg aagagttgga ggaaacccgg aagttgttga tcgcatggag caaacattcc
     2461 agggtccccg gcgagccgaa ccgtctggac aaagaagaaa cgtttgcacc gcagcagctc
     2521 caacatatcc aaaacgtcaa agacgaccgc ttgaagcaga tggcaaatct gctcgtgatg
     2581 accgccttgg gctacaagta cgatgaggcc gagaaacaat ggaaagaggc gtatccggcg
     2641 tgtcagatga ttctgttcga agatctaagc cgctaccgct ttgcgctgga ccgaccgcgt
     2701 cgcgagaaca accggttaat gaaatgggca cacagaagta tcccgcgtct ggtttatctg
     2761 caaggtgaat tgttcggcat ccaggtgggt gatgtttatt cggcgtatac tagccgtttc
     2821 catgctaaaa ctggtgcgcc aggcattcgc tgccacgcgc tgaaagaaga agacctgcaa
     2881 ccgaacagct acgtggtgaa gcaactgatt aaagacggct ttatccgtga ggaccagacc
     2941 ggttccctta agccgggtca gattgtaccg tggtctggcg gtgaactgtt cgttacctta
     3001 gccgaccgtt ccggcagccg tttggccgtg atccacgccg acatcaatgc agcgcagaat
     3061 ctgcaaaaga gattctggca acagaacacc gagatctttc gtgtgccgtg caaagtgact
     3121 acgtccggtc tgataccggc atacgacaag atgaaaaagt tattcggcaa aggctatttc
     3181 gcaaagatca accagacgga caccagcgaa gtgtacgttt gggaacacag cgctaagatg
     3241 aagggcaaaa cgaccccggc ggacccagca gaagaaggag tttttgatga gtccctgacc
     3301 gacgagatgg aagagcttga agacagccaa gagggttaca agaccctgtt ccgtgatccg
     3361 tcgggctttt tttggtcgag cgatcgctgg ctgccgcaga aagagttctg gttttgggtt
     3421 aaacgccgta tcgagaagaa actgcgcgag cagctccagc tcgagcacca ccaccaccac
     3481 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     3541 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     3601 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     3661 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     3721 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3781 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3841 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3901 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3961 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     4021 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     4081 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     4141 ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca
     4201 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     4261 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     4321 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     4381 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     4441 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     4501 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     4561 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     4621 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     4681 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     4741 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     4801 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     4861 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     4921 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc
     4981 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     5041 ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     5101 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     5161 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     5221 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     5281 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     5341 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     5401 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     5461 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     5521 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5581 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5641 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5701 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5761 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     5821 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     5881 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     5941 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     6001 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     6061 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     6121 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     6181 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     6241 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     6301 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     6361 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     6421 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     6481 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     6541 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     6601 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     6661 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     6721 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     6781 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     6841 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     6901 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     6961 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     7021 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     7081 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     7141 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     7201 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     7261 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     7321 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     7381 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     7441 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     7501 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     7561 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     7621 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     7681 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     7741 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     7801 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     7861 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     7921 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     7981 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     8041 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     8101 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     8161 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     8221 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     8281 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     8341 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     8401 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     8461 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     8521 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     8581 gagatctcga tcccgcgaaa t
//
