LOCUS       sequence-322667-        6997 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Bsu large fragment for affinity purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6997)
  AUTHORS   Patchsung M, Homchan A, Aphicho K, Suraritdechachai S, Wanitchanon 
            T, Pattama A, Sappakhaw K, Meesawat P, Wongsatit T, Athipanyasilp A,
            Jantarug K, Athipanyasilp N, Buahom J, Visanpattanasin S, 
            Niljianskul N, Chaiyen P, Tinikul R, Wichukchinda N, Mahasirimongkol
            S, Sirijatuphat R, Angkasekwinai N, Crone MA, Freemont PS, Joung J, 
            Ladha A, Abudayyeh O, Gootenberg J, Zhang F, Chewapreecha C, 
            Chanarat S, Horthongkham N, Pakotiprapha D, Uttamapinant C
  TITLE     A Multiplexed Cas13-Based Assay with Point-of-Care Attributes for 
            Simultaneous COVID-19 Diagnosis and Variant Surveillance.
  JOURNAL   CRISPR J. 2022 Nov 11. doi: 10.1089/crispr.2022.0048.
  PUBMED    36367987
REFERENCE   2  (bases 1 to 6997)
  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: "CRISPR J. 
            2022 Nov 11. doi: 10.1089/crispr.2022.0048."
COMMENT     Sequence Label: pET28a-MH6-Bsu
FEATURES             Location/Qualifiers
     source          1..6997
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(646..1728)
                     /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     1690..1709
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1729..1806)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2012..2031
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2115..2134
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2115..2133
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2134..2158
                     /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)."
     promoter        join(2167..2172,2173..2190,2191..2196)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     RBS             2216..2238
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2270..2287
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4046..4064)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4060..4107
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4144..4599
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4231..4250)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4441..4462
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4691..5506)
                     /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(4691..5506)
                     /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     5414..5433
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5498..5517
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5628..6216
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6117..6136
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6370..6387
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6402..6541
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6467..6486)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6627..6649
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6643..6834)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
       61 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
      121 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
      181 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
      241 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
      301 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
      361 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
      421 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
      481 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
      541 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
      601 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
      661 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
      721 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
      781 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
      841 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
      901 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
      961 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     1021 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     1081 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     1141 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     1201 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     1261 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     1321 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     1381 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     1441 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     1501 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     1561 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     1621 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     1681 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     1741 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     1801 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     1861 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     1921 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     1981 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     2041 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     2101 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     2161 tctagattta cactttatgc ttccggctcg tatgtttcta gagaattcaa ataattttgt
     2221 ttaactttaa gaaggagatt taaatatgaa aatcgaagaa ggtaaaggtc accatcacca
     2281 tcaccacatg gatcagagcc tggaagatat taatgttaaa accgttaccg atgtgaccag
     2341 cgatattctg gttagcccga gcgcatttgt tgttgagcag attggtgata actatcacga
     2401 agaaccgatt ctgggtttta gcattgttaa tgaaaccggt gcctatttca tcccgaaaga
     2461 tattgcagtt gaaagcgaag tgttcaaaga atgggttgaa aacgacgagc agaaaaaatg
     2521 ggtgtttgat agcaaacgtg cagttgttgc actgcgttgg cagggtattg aactgaaagg
     2581 tgcagaattt gataccctgc tggcagccta tattatcaat ccgggtaata gctatgatga
     2641 tgttgcaagc gttgccaaag attatggtct gcatattgtt agcagtgatg aaagcgttta
     2701 tggcaaaggt gcaaaacgtg ccgttccgag cgaagatgtt ctgagcgaac atctgggtcg
     2761 taaagcactg gcaattcaga gtctgcgtga aaaactggtt caagaactgg aaaataacga
     2821 tcagctggaa ctgtttgaag aattagaaat gccgctggca ctgattctgg gcgaaatgga
     2881 aagcaccggt gttaaagttg atgttgatcg tctgaaacgt atgggtgaag aactgggtgc
     2941 caaactgaaa gaatatgaag aaaaaatcca cgagattgcc ggtgaaccgt ttaatatcaa
     3001 tagcccgaaa cagctgggtg tgatcctgtt tgaaaaaatt ggtctgccgg ttgtgaaaaa
     3061 aaccaaaacc ggttatagca ccagcgcaga tgtgctggaa aaattagcag ataaacatga
     3121 catcgtggac tacattctgc agtatcgtca gattggtaaa ctgcagagca cctatattga
     3181 aggtctgctg aaagttaccc gtccggatag ccataaagtt catacccgtt ttaatcaggc
     3241 actgacccag accggtcgtc tgagcagcac cgatccgaat ctgcagaata ttccgattcg
     3301 tctggaagaa ggtcgtaaaa ttcgtcaggc atttgtgccg agcgaaaaag attggctgat
     3361 ttttgcagca gattatagcc agattgaact gcgtgttctg gcacatatta gcaaagatga
     3421 aaatctgatc gaagccttca ccaacgatat ggatattcat accaaaacag ccatggatgt
     3481 gtttcatgtt gcgaaagatg aagttaccag cgcaatgcgt cgtcaggcca aagcagttaa
     3541 ttttggtatt gtgtatggca tcagcgatta tggcctgagc cagaatctgg gtattacccg
     3601 taaagaagca ggcgcattta ttgatcgcta tctggaaagt tttcagggcg ttaaagcata
     3661 catggaagat agcgttcaag aggcaaaaca gaaaggttat gttaccacac tgatgcatcg
     3721 tcgtcgttat attccggaac tgaccagccg taatttcaat attcgtagct ttgcagaacg
     3781 taccgcaatg aataccccga ttcagggtag cgcagcagat atcatcaaaa aagcaatgat
     3841 tgacatggca gcgaagctga aagaaaaaca gctgaaagca cgtctgctgc tgcaagtgca
     3901 tgatgaactg atctttgaag caccgaaaga agaaatcgag atcctggaaa agctggttcc
     3961 ggaagttatg gaacatgcac tggcactgga tgttccgctg aaagtggatt ttgcaagcgg
     4021 tccgagctgg tatgatgcaa aataaccgct gagcaataac tagcataacc ccttggggcc
     4081 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     4141 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     4201 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     4261 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     4321 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     4381 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     4441 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     4501 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     4561 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     4621 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4681 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     4741 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     4801 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     4861 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     4921 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     4981 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     5041 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     5101 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     5161 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     5221 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     5281 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     5341 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     5401 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     5461 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     5521 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     5581 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5641 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     5701 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     5761 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     5821 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     5881 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     5941 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6001 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6061 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6121 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6181 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6241 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     6301 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     6361 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     6421 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     6481 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     6541 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     6601 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     6661 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     6721 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     6781 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     6841 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     6901 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     6961 acaactggcg gtatggatgc ggcgggacca gagaaaa
//
