LOCUS       sequence-212075-        5644 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Used for expression of protein in E. coli as PreScission cleavable 
            N-terminual 8His and MBP tag fusions and with C-terminal Avi-tag .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5644)
  TITLE     Hyvonen Lab pOP plasmids
REFERENCE   2  (bases 1 to 5644)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pOP5MP
FEATURES             Location/Qualifiers
     source          1..5644
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    19..43
                     /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             56..78
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             98..121
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             128..1228
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGIDAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1202..1225
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1268..1291
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1328..1372
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     promoter        complement(1389..1407)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1390..1407)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1465..1483)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1479..1526
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1762..1783)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1933..3015)
                     /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
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     2977..2996
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3016..3093)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     3239..3258
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3414..3433)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3533..3555
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3593..3611)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3679..3783
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3784..3852,3853..4644)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCHTLLSRIDAGQEQLGRRARYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      4815..5403
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5304..5323
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5557..5574
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tggatctcat caccatcacc atcaccatca
      121 cactagtatg aaaatcgaag aaggtaaact ggtaatctgg attaacggcg ataaaggcta
      181 taacggtctc gctgaagtcg gtaagaaatt cgagaaagat accggaatta aagtcaccgt
      241 tgagcatccg gataaactgg aagagaaatt cccacaggtt gcggcaactg gcgatggccc
      301 tgacattatc ttctgggcac acgaccgctt tggtggctac gctcaatctg gcctgttggc
      361 tgaaatcacc ccggacaaag cgttccagga caagctgtat ccgtttacct gggatgccgt
      421 acgttacaac ggcaagctga ttgcttaccc gatcgctgtt gaagcgttat cgctgattta
      481 taacaaagat ctgctgccga acccgccaaa aacctgggaa gagatcccgg cgctggataa
      541 agaactgaaa gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac cgtacttcac
      601 ctggccgctg attgctgctg acgggggtta tgcgttcaag tatgaaaacg gcaagtacga
      661 cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct tcctggttga
      721 cctgattaaa aacaaacaca tgaatgcaga caccgattac tccatcgcag aagctgcctt
      781 taataaaggc gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca acatcgacac
      841 cagcaaagtg aattatggtg taacggtact gccgaccttc aagggtcaac catccaaacc
      901 gttcgttggc gtgctgagcg caggtattga cgccgccagt ccgaacaaag agctggcaaa
      961 agagttcctc gaaaactatc tgctgactga tgaaggtctg gaagcggtta ataaagacaa
     1021 accgctgggt gccgtagcgc tgaagtctta cgaggaagag ttggcgaaag atccacgtat
     1081 tgccgccaca atggaaaacg cccagaaagg tgaaatcatg ccgaacatcc cgcagatgtc
     1141 cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc gccagcggtc gtcagactgt
     1201 cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg accggtagtg gcaccagtgg
     1261 gtcgacactg gaagttctgt ttcagggccc aggatccatg gaattcgcgg ccgccctagg
     1321 ctcgagcgga ctgaatgaca ttttcgaagc acagaagatc gaatggcatg aagcctaagc
     1381 ttgagtattc tatagtgtca cctaaatccc agcttgatcc ggctgctaac aaagcccgaa
     1441 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     1501 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga taacctggcg
     1561 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     1621 atggaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     1681 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     1741 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     1801 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccacagct tgcatgcctg
     1861 caggtcggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta acttacatta
     1921 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     1981 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     2041 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     2101 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttga
     2161 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     2221 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     2281 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     2341 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     2401 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     2461 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     2521 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     2581 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     2641 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     2701 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     2761 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     2821 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     2881 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     2941 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     3001 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     3061 accgcgaaag gttttgcacc attcgatggt gtcgggtacc gagctcgaat ttacgtgaac
     3121 catcacccaa atcaagtttt ttgcggtcga ggtgccgtaa agctctaaat cggaacccta
     3181 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     3241 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     3301 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcgcct gatgcggtat
     3361 tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc
     3421 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     3481 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     3541 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     3601 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     3661 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     3721 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     3781 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     3841 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     3901 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     3961 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtca tacactatta
     4021 tcccgtattg acgccgggca agagcaactc ggtcgccggg cgcggtattc tcagaatgac
     4081 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     4141 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     4201 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     4261 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     4321 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     4381 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     4441 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     4501 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     4561 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     4621 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     4681 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     4741 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     4801 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     4861 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     4921 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     4981 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     5041 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     5101 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     5161 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     5221 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5281 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5341 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5401 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5461 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     5521 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     5581 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     5641 tgat
//
