LOCUS       40924_18626        8232 bp DNA     circular SYN 13-MAY-2021
DEFINITION  pET vector for IPTG-inducible, T7-based expression of the 
            exonuclease deficient version 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, Ellington AD
  TITLE     A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for 
            reagent production
  JOURNAL   bioRxiv
REFERENCE   2  (bases 1 to 8232)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8232)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8232
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      40..126
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      223..309
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      406..492
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      589..675
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      complement(775..818)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     promoter        993..1011
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1012..1036
                     /label=lac operator
                     /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             1051..1073
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     RBS             3009..3017
                     /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             3418..3435
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      3516..3563
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        3623..3727
                     /label=AmpR promoter
     CDS             3728..4585
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      complement(4630..5143)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     rep_origin      5254..5842
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5996..6013
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(6035..6054)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     6195..6217
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6214..6402)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(6925..6946)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(6962..8041)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARPRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(8042..8119)
                     /label=lacI promoter
ORIGIN
        1 ctttcgtctt caagaattaa ttcccaattc cccaggcatc aaataaaacg aaaggctcag
       61 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
      121 acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca
      181 ggacgcccgc cataaactgc caggaattaa ttccccaggc atcaaataaa acgaaaggct
      241 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
      301 aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg
      361 gcaggacgcc cgccataaac tgccaggaat taattcccca ggcatcaaat aaaacgaaag
      421 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
      481 agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg
      541 cgggcaggac gcccgccata aactgccagg aattaattcc ccaggcatca aataaaacga
      601 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
      661 ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg
      721 tggcgggcag gacgcccgcc ataaactgcc aggaattaat tccccaggca tcaaataaaa
      781 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
      841 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga
      901 gggtggcggg caggacgccc gccataaact gccaggaatt ggggatcgga attaattccc
      961 ggtttaaacc ggggatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
     1021 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacat
     1081 atgattcttg acacggatta catcacggaa gacggcaagc cggttatccg tattttcaag
     1141 aaagaaaacg gcgaattcaa gattgaatac gatcggacat ttgaaccgta cctgtacgct
     1201 ctcctcaagg atgatagcgc aatcgaagaa gtgaaaaaaa tcaccgcaga gcggcatggc
     1261 acagtggtaa cagttaagcg ggtcgagaaa gtgcagaaga agttcttagg ccggccagtc
     1321 gaagtatgga aattatactt cacacatcca caggacgttc cggcgatcat ggataagatt
     1381 cgggagcatc cggcggtaat cgatatctat gaatacgata ttccgttcgc tattcgctac
     1441 cttattgaca aaggtttagt tccaatggag ggtgatgagg aacttaaact gttagcattc
     1501 gatatcgaaa cactttatca cgaaggtgaa gagtttgccg aaggtccgat tttaatgatc
     1561 tcgtacgccg atgaagaagg cgcacgcgta attacgtgga aaaatgtgga cctcccgtac
     1621 gtagacgtag tgagcactga gcgcgagatg attaaacgtt tccttcgggt agtaaaagaa
     1681 aaagacccag acgtgctgat tacgtatgac ggcgacaact ttgattttgc ctatctcaag
     1741 aagcgttgcg aaaagttagg cattaatttc gccctgggtc gggacggttc agagccgaaa
     1801 attcagcgga tgggcgaccg ctttgctgtg gaggtaaaag gtcgcatcca tttcgattta
     1861 tatccggtta tccggcgcac catcaacttg ccgacttaca cacttgaagc agtttacgaa
     1921 gcggtgttcg gccaaccaaa agaaaaggtt tatgccgagg agattaccac cgcatgggaa
     1981 actggcgaaa acttggagcg ggtggctcgg tattccatgg aagatgccaa ggtgacctac
     2041 gaactgggca aagagttttt accgatggaa gcacaattaa gccgccttat tggtcagtcc
     2101 ctctgggatg tgtcgcgttc ttcaacgggc aatttagtcg aatggtttct tcttcggaaa
     2161 gcgtacgagc gtaacgagct tgctccaaat aagccagacg aaaaagaatt ggctcggcgc
     2221 catcagtcac atgagggcgg ctacattaag gagccagaac ggggcttgtg ggagaacatc
     2281 gtctaccttg attttcggtc tctttatccg tctattatca tcacacataa cgtctcgcca
     2341 gataccctga accgtgaagg ctgtaaagaa tatgatgtgg caccacaggt cggccatcgt
     2401 ttttgtaaag acttcccggg cttcattcca tctcttctgg gtgatttgtt agaagagcgt
     2461 caaaagatca agaaacgtat gaaagcgaca attgacccaa ttgaacgcaa attacttgat
     2521 taccgtcagc gtgcaatcaa gatcctcgcg aactctctgt acggttatta cggctacgca
     2581 cgcgcccggt ggtattgcaa agaatgtgca gaatcagtca ttgcttgggg tcgggagtac
     2641 ctgaccatga cgattaagga aattgaggag aaatacggtt tcaaggtcat ctatagtgac
     2701 acggatggtt tctttgcaac gattccaggt gcggacgcag aaactgtaaa gaaaaaggca
     2761 atggagttct tgaagtatat taatgcgaag ttgccaggcg ccctggaatt agagtacgaa
     2821 ggtttttata agcgtggcct gttcgtgaca aagaagaaat acgcggtaat tgacgaggaa
     2881 ggcaagatca caactcgtgg cttggaaatt gttcgtcgcg attggagcga gatcgcaaag
     2941 gagacccaag ctcgtgtgtt ggaggccctc ctgaaggatg gtgacgtcga aaaagccgta
     3001 cgcatcgtta aggaggttac agagaagctt agcaagtatg aggtcccacc agagaaactt
     3061 gttattcata aacaaatcac tcgcgacctt aaagactata aggccactgg tccacacgtc
     3121 gccgtagcaa agcggcttgc ggctcggggc gtcaagattc ggccaggcac ggttattagt
     3181 tacatcgtcc tcaaaggctc aggccggatt gttgatcgcg cgattccatt tgatgaattt
     3241 gatccgacga agcataaata tgatgcggaa tattacattg aaaaacaggt tctgccggcg
     3301 gtggagcgca tcttacgtgc gttcggctat cgcaaggagg atttgcggta ccagaaaact
     3361 cgtcaagtcg gtttgagtgc ccgtctgaag ccgaaaggta ccctcgaggg atccagccat
     3421 caccatcacc atcactgata agcggccgca ttgatccggc tgctaacaaa gcccgaaagg
     3481 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     3541 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggataa ctacgtcagg
     3601 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     3661 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     3721 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     3781 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     3841 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     3901 tcgccccgaa gaacgtttcc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     3961 attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     4021 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     4081 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     4141 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     4201 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     4261 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     4321 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     4381 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     4441 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     4501 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     4561 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     4621 gattgattta ccccggttga taatcagaaa agccccaaaa acaggaagat tgtataagca
     4681 aatatttaaa ttgtaaacgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     4741 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     4801 cccgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     4861 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     4921 tcacccaaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     4981 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     5041 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     5101 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtaaaagga tctaggtgaa
     5161 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     5221 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     5281 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     5341 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     5401 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     5461 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5521 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5581 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5641 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     5701 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5761 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     5821 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5881 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     5941 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     6001 gtcagtgagc gaggaagcta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     6061 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     6121 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     6181 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     6241 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtgc agcgattcac agatgtctgc
     6301 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     6361 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     6421 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     6481 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     6541 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagccga acgccagcaa
     6601 gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     6661 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     6721 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     6781 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     6841 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     6901 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     6961 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     7021 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     7081 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     7141 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     7201 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc
     7261 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     7321 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     7381 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     7441 agccagccag acgcggacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     7501 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     7561 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     7621 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     7681 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     7741 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     7801 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     7861 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     7921 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     7981 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     8041 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     8101 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     8161 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     8221 cggcatgccg cc
//
