LOCUS       sequence-292639-        8232 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET vector for IPTG-inducible, T7-based expression of RTX bearing a 
            C-terminal 6xHis tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8232)
  AUTHORS   Bhadra S, Maranhao AC, Paik I, Ellington AD
  TITLE     A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for 
            reagent production
  JOURNAL   BioProtocol 2021
REFERENCE   2  (bases 1 to 8232)
  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: "BioProtocol
            2021"
COMMENT     Sequence Label: pET_RTX-6xHis
FEATURES             Location/Qualifiers
     source          1..8232
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      86..172
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      269..355
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      452..538
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      635..721
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      818..904
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     1039..1058
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1039..1057
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1058..1082
                     /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             1097..1119
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     RBS             3055..3063
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             3464..3481
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3548..3566)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3562..3609
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        3669..3773
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3774..3842,3843..4634)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3992..4011)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(4676..5189)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4871..4892)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5083..5102
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      5300..5888
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5789..5808
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6042..6059
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(6081..6100)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6241..6263
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6257..6448)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7005..8087)
                     /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
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARPRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     8049..8068
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8088..8165)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gttgagcacc gccgccgcaa ggaatggtgc atgccggcat gccgcccttt cgtcttcaag
       61 aattaattcc caattcccca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
      121 ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg
      181 agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata
      241 aactgccagg aattaattcc ccaggcatca aataaaacga aaggctcagt cgaaagactg
      301 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc
      361 gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc
      421 ataaactgcc aggaattaat tccccaggca tcaaataaaa cgaaaggctc agtcgaaaga
      481 ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc
      541 gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc
      601 gccataaact gccaggaatt aattccccag gcatcaaata aaacgaaagg ctcagtcgaa
      661 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
      721 tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg
      781 cccgccataa actgccagga attaattccc caggcatcaa ataaaacgaa aggctcagtc
      841 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac
      901 aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg
      961 acgcccgcca taaactgcca ggaattgggg atcggaatta attcccggtt taaaccgggg
     1021 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
     1081 cccctctaga aataattttg tttaacttta agaaggagat atacatatga ttcttgacac
     1141 ggattacatc acggaagacg gcaagccggt tatccgtatt ttcaagaaag aaaacggcga
     1201 attcaagatt gaatacgatc ggacatttga accgtacctg tacgctctcc tcaaggatga
     1261 tagcgcaatc gaagaagtga aaaaaatcac cgcagagcgg catggcacag tggtaacagt
     1321 taagcgggtc gagaaagtgc agaagaagtt cttaggccgg ccagtcgaag tatggaaatt
     1381 atacttcaca catccacagg acgttccggc gatcatggat aagattcggg agcatccggc
     1441 ggtaatcgat atctatgaat acgatattcc gttcgctatt cgctacctta ttgacaaagg
     1501 tttagttcca atggagggtg atgaggaact taaactgtta gcattcgata tcgaaacact
     1561 ttatcacgaa ggtgaagagt ttgccgaagg tccgatttta atgatctcgt acgccgatga
     1621 agaaggcgca cgcgtaatta cgtggaaaaa tgtggacctc ccgtacgtag acgtagtgag
     1681 cactgagcgc gagatgatta aacgtttcct tcgggtagta aaagaaaaag acccagacgt
     1741 gctgattacg tataacggcg acaactttga ttttgcctat ctcaagaagc gttgcgaaaa
     1801 gttaggcatt aatttcgccc tgggtcggga cggttcagag ccgaaaattc agcggatggg
     1861 cgaccgcttt gctgtggagg taaaaggtcg catccatttc gatttatatc cggttatccg
     1921 gcgcaccatc aacttgccga cttacacact tgaagcagtt tacgaagcgg tgttcggcca
     1981 accaaaagaa aaggtttatg ccgaggagat taccaccgca tgggaaactg gcgaaaactt
     2041 ggagcgggtg gctcggtatt ccatggaaga tgccaaggtg acctacgaac tgggcaaaga
     2101 gtttttaccg atggaagcac aattaagccg ccttattggt cagtccctct gggatgtgtc
     2161 gcgttcttca acgggcaatt tagtcgaatg gtttcttctt cggaaagcgt acgagcgtaa
     2221 cgagcttgct ccaaataagc cagacgaaaa agaattggct cggcgccatc agtcacatga
     2281 gggcggctac attaaggagc cagaacgggg cttgtgggag aacatcgtct accttgattt
     2341 tcggtctctt tatccgtcta ttatcatcac acataacgtc tcgccagata ccctgaaccg
     2401 tgaaggctgt aaagaatatg atgtggcacc acaggtcggc catcgttttt gtaaagactt
     2461 cccgggcttc attccatctc ttctgggtga tttgttagaa gagcgtcaaa agatcaagaa
     2521 acgtatgaaa gcgacaattg acccaattga acgcaaatta cttgattacc gtcagcgtgc
     2581 aatcaagatc ctcgcgaact ctctgtacgg ttattacggc tacgcacgcg cccggtggta
     2641 ttgcaaagaa tgtgcagaat cagtcattgc ttggggtcgg gagtacctga ccatgacgat
     2701 taaggaaatt gaggagaaat acggtttcaa ggtcatctat agtgacacgg atggtttctt
     2761 tgcaacgatt ccaggtgcgg acgcagaaac tgtaaagaaa aaggcaatgg agttcttgaa
     2821 gtatattaat gcgaagttgc caggcgccct ggaattagag tacgaaggtt tttataagcg
     2881 tggcctgttc gtgacaaaga agaaatacgc ggtaattgac gaggaaggca agatcacaac
     2941 tcgtggcttg gaaattgttc gtcgcgattg gagcgagatc gcaaaggaga cccaagctcg
     3001 tgtgttggag gccctcctga aggatggtga cgtcgaaaaa gccgtacgca tcgttaagga
     3061 ggttacagag aagcttagca agtatgaggt cccaccagag aaacttgtta ttcataaaca
     3121 aatcactcgc gaccttaaag actataaggc cactggtcca cacgtcgccg tagcaaagcg
     3181 gcttgcggct cggggcgtca agattcggcc aggcacggtt attagttaca tcgtcctcaa
     3241 aggctcaggc cggattgttg atcgcgcgat tccatttgat gaatttgatc cgacgaagca
     3301 taaatatgat gcggaatatt acattgaaaa acaggttctg ccggcggtgg agcgcatctt
     3361 acgtgcgttc ggctatcgca aggaggattt gcggtaccag aaaactcgtc aagtcggttt
     3421 gagtgcccgt ctgaagccga aaggtaccct cgagggatcc agccatcacc atcaccatca
     3481 ctgataagcg gccgcattga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     3541 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     3601 ggttttttgc tgaaaggagg aactatatcc ggataactac gtcaggtggc acttttcggg
     3661 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     3721 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     3781 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     3841 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     3901 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     3961 gtttcccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg
     4021 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     4081 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     4141 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     4201 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     4261 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     4321 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     4381 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     4441 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     4501 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     4561 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     4621 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttacccc
     4681 ggttgataat cagaaaagcc ccaaaaacag gaagattgta taagcaaata tttaaattgt
     4741 aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     4801 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagcccg agatagggtt
     4861 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     4921 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac ccaaatcaag
     4981 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     5041 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     5101 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     5161 cgcgcttaat gcgccgctac agggcgcgta aaaggatcta ggtgaagatc ctttttgata
     5221 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     5281 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     5341 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     5401 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     5461 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     5521 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     5581 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     5641 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     5701 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     5761 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     5821 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     5881 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     5941 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     6001 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     6061 aagctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     6121 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     6181 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     6241 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     6301 ggtaaagctc atcagcgtgg tcgtgcagcg attcacagat gtctgcctgt tcatccgcgt
     6361 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     6421 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     6481 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     6541 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     6601 accagagaaa aatcactcag ggtcaatgcc agccgaacgc cagcaagacg tagcccagcg
     6661 cgtcggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     6721 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     6781 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     6841 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     6901 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     6961 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     7021 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     7081 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     7141 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     7201 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     7261 gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg
     7321 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     7381 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     7441 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     7501 ggacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     7561 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     7621 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     7681 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     7741 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     7801 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     7861 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     7921 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     7981 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     8041 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     8101 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     8161 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     8221 taggttgagg cc
//
