LOCUS       sequence-247625-        6355 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses untagged human rhinovirus 3C protease in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6355)
  AUTHORS   Raran-Kurussi S, Waugh DS
  TITLE     A dual protease approach for expression and affinity purification of
            recombinant proteins.
  JOURNAL   Anal Biochem. 2016 Apr 19;504:30-37. doi: 10.1016/j.ab.2016.04.006.
  PUBMED    27105777
REFERENCE   2  (bases 1 to 6355)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Anal 
            Biochem."; date: "2016-04-19"; volume: "504:30-37. doi"; pages: " 
            10.1016/j.ab.2016.04.006"
COMMENT     Sequence Label: pSRK2706
FEATURES             Location/Qualifiers
     source          1..6355
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..46
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     130..149
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        130..148
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    149..173
                     /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)."
     protein_bind    214..238
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(812..836)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             856..897
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             907..924
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(981..999)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      995..1042
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1217..1222,1223..1239,1240..1245))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1276..1294)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1362..1466
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1467..1535,1536..2327)
                     /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(1685..1704)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2498..3086
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2987..3006
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3240..3257
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3272..3411
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3337..3356)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3497..3519
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3513..3704)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5016..6098)
                     /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     6060..6079
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6099..6176)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
       61 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
      121 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
      181 aaataatttt gtttaacttt aagaaggaat atcacaagtt tgtacaaaaa agcaggcttt
      241 aagaaggaga tatacatatg ggaccaaaca cagaatttgc actatccctg ttaaggaaaa
      301 acataatgac tataacaacc tcaaagggag agttcacagg gttaggcata catgatcgtg
      361 tctgtgtgat acccacacac gcacagcctg gtgatgatgt actagtaaat ggtcagaaaa
      421 ttagagttaa ggataagtac aaattagtag atccagagaa cattaatcta gagcttacag
      481 tgttgacttt agatagaaat gaaaaattca gagatatcag gggatttata tcagaagatc
      541 tagaaggtgt ggatgccact ttggtagtac attcaaataa ctttaccaac actatcttag
      601 aagttggccc tgtaacaatg gcaggactta ttaatttgag tagcaccccc actaacagaa
      661 tgattcgtta tgattatgca acaaaaactg ggcagtgtgg aggtgtgctg tgtgctactg
      721 gtaagatctt tggtattcat gttggcggta atggaagaca aggattttca gctcaactta
      781 aaaaacaata ttttgtagag aaacaataat aacccagctt tcttgtacaa agtggtgatc
      841 aattcgaagc ttgaaggtaa gcctatccct aaccctctcc tcggtctcga ttctacgcgt
      901 accggtcatc atcaccatca ccattgagtt tgatccggct gctaacaaag cccgaaagga
      961 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     1021 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatatc ccgcaagagg
     1081 cccggcagta ccggcataac caagcctatg cctacagcat ccagggtgac ggtgccgagg
     1141 atgacgatga gcgcattgtt agatttcata cacggtgcct gactgcgtta gcaatttaac
     1201 tgtgataaac taccgcatta aagcttatcg atgataagct gtcaaacatg agaattaatt
     1261 cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     1321 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     1381 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     1441 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     1501 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     1561 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     1621 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     1681 ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc
     1741 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     1801 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     1861 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     1921 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     1981 caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat
     2041 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     2101 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     2161 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     2221 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     2281 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     2341 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     2401 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     2461 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2521 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     2581 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     2641 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     2701 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     2761 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     2821 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     2881 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     2941 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     3001 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     3061 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     3121 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     3181 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     3241 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     3301 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     3361 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     3421 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     3481 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     3541 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     3601 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     3661 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     3721 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     3781 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     3841 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     3901 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     3961 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     4021 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     4081 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     4141 aacgacagga gcacgatcat gcgcacccgt ggccaggacc caacgctgcc cgagatgcgc
     4201 cgcgtgcggc tgctggagat ggcggacgcg atggatatgt tctgccaagg gttggtttgc
     4261 gcattcacag ttctccgcaa gaattgattg gctccaattc ttggagtggt gaatccgtta
     4321 gcgaggtgcc gccggcttcc attcaggtcg aggtggcccg gctccatgca ccgcgacgca
     4381 acgcggggag gcagacaagg tatagggcgg cgcctacaat ccatgccaac ccgttccatg
     4441 tgctcgccga ggcggcataa atcgccgtga cgatcagcgg tccaatgatc gaagttaggc
     4501 tggtaagagc cgcgagcgat ccttgaagct gtccctgatg gtcgtcatct acctgcctgg
     4561 acagcatggc ctgcaacgcg ggcatcccga tgccgccgga agcgagaaga atcataatgg
     4621 ggaaggccat ccagcctcgc gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg
     4681 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     4741 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     4801 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     4861 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     4921 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     4981 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5101 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     5161 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     5221 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     5281 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     5341 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     5401 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     5461 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     5521 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     5581 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     5641 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     5701 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     5761 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     5821 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     5881 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     5941 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     6001 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     6061 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     6121 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     6181 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     6241 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     6301 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccg
//
