LOCUS       sequence-322668-        6121 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses T4 gp32 for bacterial expression and affinity 
            purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6121)
  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 6121)
  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-gp32-H6
FEATURES             Location/Qualifiers
     source          1..6121
                     /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             3149..3166
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3170..3188)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3184..3231
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3268..3723
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3355..3374)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3565..3586
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3815..4630)
                     /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(3815..4630)
                     /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     4538..4557
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4622..4641
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4752..5340
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5241..5260
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5494..5511
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5526..5665
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5591..5610)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5751..5773
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5767..5958)
                     /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 taaatatgtt caaacgtaaa agcaccgcag aactggcagc
     2281 acagatggca aaactgaatg gtaataaagg ttttagcagc gaggataaag gtgagtggaa
     2341 actgaaactg gataatgccg gtaatggtca ggcagttatt cgttttctgc cgagcaaaaa
     2401 tgatgaacag gcaccgtttg caattctggt gaatcatggt ttcaaaaaga acggcaaatg
     2461 gtacattgaa acctgtagca gcacccatgg tgattatgat agctgtccgg tttgtcagta
     2521 tatcagcaaa aacgatctgt acaacaccga taacaaagaa tacagcctgg ttaaacgcaa
     2581 aaccagctat tgggcaaata ttctggttgt taaagatccg gcagcaccgg aaaatgaagg
     2641 taaagttttc aaatatcgct ttggcaagaa gatctgggat aaaatcaatg ccatgattgc
     2701 cgttgatgtg gaaatgggtg aaacaccggt tgatgttacc tgtccgtggg aaggtgcaaa
     2761 ttttgttctg aaagttaaac aggtgagcgg cttcagcaat tatgatgaaa gcaaatttct
     2821 gaaccagagc gccattccga atatcgatga tgaatcattt cagaaagaac tgttcgagca
     2881 gatggttgat ctgagcgaaa tgaccagcaa agacaaattc aaaagcttcg aagaactgaa
     2941 cacgaaattt ggtcaggtta tgggcaccgc agttatgggt ggtgcagcag caaccgcagc
     3001 aaaaaaagca gataaagttg cagatgatct ggatgccttt aacgtggatg attttaacac
     3061 caaaaccgag gatgatttca tgagcagcag cagcggtagc agcagctcag cagatgatac
     3121 cgatctggac gatctgctga atgatctgca ccatcaccat caccactaac cgctgagcaa
     3181 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     3241 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     3301 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3361 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3421 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3481 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3541 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3601 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     3661 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     3721 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     3781 acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat
     3841 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     3901 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     3961 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     4021 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     4081 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     4141 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     4201 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     4261 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     4321 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     4381 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     4441 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     4501 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     4561 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     4621 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
     4681 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4741 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4801 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4861 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     4921 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4981 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     5041 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     5101 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     5161 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     5221 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     5281 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     5341 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     5401 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     5461 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     5521 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     5581 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     5641 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     5701 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     5761 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     5821 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     5881 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     5941 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     6001 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     6061 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     6121 a
//
