LOCUS       Exported                7473 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Bacterial expression of the Bst Large Fragment.
ACCESSION   .
VERSION     .
KEYWORDS    pET15b_Bst-LF
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7473)
  TITLE     COVID-19 Diagnostic Toolkit Enzymes
REFERENCE   2  (bases 1 to 7473)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7473
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     14..33
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     117..136
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        117..135
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    136..160
                     /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             175..197
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             216..233
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             243..260
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(2090..2108)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2104..2151
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(2326..2354)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2381..2399)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2467..2571
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2572..3432
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2790..2809)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3603..4191
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4092..4111
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4345..4362
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4377..4516
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4442..4461)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4602..4624
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4618..4809)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6121..7203)
                     /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     7165..7184
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7204..7281)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
       61 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat
      121 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
      181 taactttaag aaggagatat accatgggca gcagccatca tcatcatcat cacagcagcg
      241 gcctggtgcc gcgcggcagc catagccata tgaccgacga aggcgaaaaa ccgctggcgg
      301 gtatggactt tgcgatcgct gatagcgtta ccgacgagat gctggcagat aaagcagcgc
      361 tagtagttga agttgtgggc gataactacc atcatgcgcc gatcgtgggt atcgctttgg
      421 cgaacgagcg cggccgtttt ttcctgcgcc cggaaaccgc actggcggac ccgaagtttc
      481 tggcttggct gggcgacgaa acgaaaaaaa agactatgtt cgattctaaa cgtgcagcgg
      541 tggctctcaa atggaaaggt atcgaattgc gcggtgttgt gttcgacctg ctgctggctg
      601 cgtacctgct cgatcctgct caggctgcgg gtgacgtggc agctgtagcg aaaatgcacc
      661 agtacgaagc cgtacgctct gatgaagcgg tttatggtaa aggtgcgaaa cgtaccgtgc
      721 cagacgaacc aaccctggcg gaacacctgg ctcgcaaagc agccgctatc tgggccctgg
      781 aggagcctct gatggatgaa cttcgccgca acgaacagga ccgtctgctc acagaactgg
      841 aacagccgtt ggcgggtatt ctggcaaaca tggagttcac tggtgtaaaa gtggacacta
      901 agcgcctgga acaaatgggt gccgaactga ctgagcagct tcaggcagtt gaacgccgca
      961 tctacgaact ggcgggtcag gagtttaaca tcaactctcc taaacagctg ggcaccgtgt
     1021 tgttcgacaa actgcaactg ccggtgttga agaaaacgaa aaccggctac agcacctccg
     1081 ccgatgttct ggaaaaactg gcaccgcatc acgaaatcgt tgaacacatc ctgcactacc
     1141 gtcagctggg taaactgcaa tctacctaca tcgagggctt actgaaagta gtgcacccgg
     1201 tgaccggtaa agtgcacact atgttcaacc aggctctgac gcagaccggc cgcctgtcct
     1261 ccgttgaacc gaacctgcaa aacatcccca tccgtctgga agagggccgt aagatccgtc
     1321 aggcattcgt gccgtctgaa ccggactggc tgattttcgc tgctgattac tctcaaatcg
     1381 aactgcgtgt tcttgctcac attgcggagg acgataacct gattgaagca ttccgtcgtg
     1441 gtctggacat tcacactaaa acggctatgg acattttcca tgtttccgaa gaagatgtga
     1501 ccgctaatat gcgtcgtcag gctaaagcgg taaactttgg tatcgtatac ggtatctccg
     1561 attacggcct ggcacagaac ctgaacatca cccgcaaaga agcggctgag tttatcgaac
     1621 gttatttcgc ctcgttcccc ggtgttaaac agtacatgga taacatcgtt caagaagcta
     1681 aacagaaggg ttatgtgacc accctgctgc accgtcgtcg ttacctgccg gacatcacct
     1741 cccgtaactt caacgtgcgt tctttcgccg aacgtacggc tatgaatacc cctatccagg
     1801 gcagcgcggc ggacatcatc aaaaaagcca tgatcgacct gagcgtgcgc ctgcgcgaag
     1861 aacgtttgca ggctcgcctg cttctgcaag tgcacgatga gctgatccta gaagctccaa
     1921 aagaggaaat tgaacgtttg tgccgtctgg ttccggaagt tatggaacag gctgtcgcac
     1981 tgcgtgttcc gcttaaagtt gattaccatt acggtcccac ttggtacgac gccaagtgag
     2041 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     2101 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     2161 ggaactatat ccggatatcc cgcaagaggc ccggcagtac cggcataacc aagcctatgc
     2221 ctacagcatc cagggtgacg gtgccgagga tgacgatgag cgcattgtta gatttcatac
     2281 acggtgcctg actgcgttag caatttaact gtgataaact accgcattaa agcttatcga
     2341 tgataagctg tcaaacatga gaattcttga agacgaaagg gcctcgtgat acgcctattt
     2401 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     2461 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     2521 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     2581 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     2641 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     2701 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     2761 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac
     2821 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     2881 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     2941 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     3001 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     3061 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca
     3121 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     3181 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     3241 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     3301 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     3361 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     3421 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     3481 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     3541 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     3601 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     3661 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     3721 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     3781 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     3841 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     3901 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     3961 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4021 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4081 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4141 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4201 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4261 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     4321 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     4381 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     4441 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     4501 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     4561 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     4621 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     4681 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     4741 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     4801 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     4861 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     4921 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     4981 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     5041 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     5101 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     5161 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     5221 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggcca
     5281 ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga
     5341 tatgttctgc caagggttgg tttgcgcatt cacagttctc cgcaagaatt gattggctcc
     5401 aattcttgga gtggtgaatc cgttagcgag gtgccgccgg cttccattca ggtcgaggtg
     5461 gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga caaggtatag ggcggcgcct
     5521 acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc
     5581 agcggtccaa tgatcgaagt taggctggta agagccgcga gcgatccttg aagctgtccc
     5641 tgatggtcgt catctacctg cctggacagc atggcctgca acgcgggcat cccgatgccg
     5701 ccggaagcga gaagaatcat aatggggaag gccatccagc ctcgcgtcgc gaacgccagc
     5761 aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     5821 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     5881 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     5941 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     6001 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     6061 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     6121 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     6181 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     6241 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     6301 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     6361 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
     6421 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     6481 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     6541 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     6601 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     6661 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     6721 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     6781 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     6841 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     6901 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     6961 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     7021 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     7081 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     7141 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     7201 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     7261 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     7321 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     7381 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     7441 aacaagcgct catgagcccg aagtggcgag ccc
//
