LOCUS       sequence-220250-        5639 bp    DNA     circular SYN 15-DEC-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 5639)
  TITLE     Hyvonen Lab pOP plasmids
REFERENCE   2  (bases 1 to 5639)
  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"
COMMENT     Sequence Label: pOP5MP
FEATURES             Location/Qualifiers
     source          1..5639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     57..76
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        57..75
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    75..99
                     /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             112..134
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             154..177
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             184..1284
                     /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     1258..1281
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1324..1347
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1384..1428
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     promoter        complement(1445..1463)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1446..1463)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1521..1539)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1535..1582
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1818..1839)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1989..3071)
                     /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     3033..3052
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3072..3149)
                     /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     3292..3311
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3465..3484)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3584..3606
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3644..3662)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3730..3834
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3835..3903,3904..4695)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4053..4072)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4866..5454
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5355..5374
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5608..5625
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctgattaat
       61 acgactcact atagggaatt gtgagcggat aacaattcct ctagaaataa ttttgtttaa
      121 ctttaagaag gagatatatc catgaatgga tctcatcacc atcaccatca ccatcacact
      181 agtatgaaaa tcgaagaagg taaactggta atctggatta acggcgataa aggctataac
      241 ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag
      301 catccggata aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac
      361 attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa
      421 atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt
      481 tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac
      541 aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa
      601 ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg
      661 ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt
      721 aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg
      781 attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat
      841 aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc
      901 aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc
      961 gttggcgtgc tgagcgcagg tattgacgcc gccagtccga acaaagagct ggcaaaagag
     1021 ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg
     1081 ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc
     1141 gccacaatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct
     1201 ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat
     1261 gaagccctga aagacgcgca gactaattcg agctcgaccg gtagtggcac cagtgggtcg
     1321 acactggaag ttctgtttca gggcccagga tccatggaat tcgcggccgc cctaggctcg
     1381 agcggactga atgacatttt cgaagcacag aagatcgaat ggcatgaagc ctaagcttga
     1441 gtattctata gtgtcaccta aatcccagct tgatccggct gctaacaaag cccgaaagga
     1501 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     1561 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggataac ctggcgtaat
     1621 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     1681 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     1741 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     1801 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     1861 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc acagcttgca tgcctgcagg
     1921 tcggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactt acattaattg
     1981 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     2041 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     2101 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     2161 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     2221 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     2281 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     2341 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     2401 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     2461 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     2521 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     2581 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     2641 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     2701 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     2761 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     2821 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     2881 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     2941 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     3001 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     3061 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     3121 cgaaaggttt tgcaccattc gatggtgtcg ggtaccgagc tcgaattgtg aaccatcacc
     3181 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     3241 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     3301 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     3361 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc tgatgcggta ttttctcctt
     3421 acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat
     3481 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     3541 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     3601 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     3661 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     3721 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     3781 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     3841 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     3901 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     3961 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     4021 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     4081 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     4141 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     4201 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     4261 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     4321 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     4381 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     4441 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     4501 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     4561 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     4621 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     4681 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     4741 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     4801 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     4861 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     4921 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4981 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     5041 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     5101 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     5161 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     5221 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     5281 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     5341 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     5401 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     5461 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     5521 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     5581 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcg
//
