LOCUS       sequence-220212-        5639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as PreScission cleavable 
            N-terminal Avi-His8-tag and MBP fusions.
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: pOP3MP
FEATURES             Location/Qualifiers
     source          1..5639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     77..96
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        77..95
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    95..119
                     /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             132..154
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             168..212
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             222..245
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             252..1352
                     /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     1326..1349
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1392..1415
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        complement(1465..1483)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1466..1483)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1541..1559)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1555..1602
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1838..1859)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2009..3091)
                     /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     3053..3072
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3092..3169)
                     /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     3312..3331
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3485..3504)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3604..3626
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3664..3682)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3750..3854
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3855..3923,3924..4715)
                     /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(4073..4092)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4886..5474
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5375..5394
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(5628..5639,1..6)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
       61 cattaatgca gctgattaat acgactcact atagggaatt gtgagcggat aacaattcct
      121 ctagaaataa ttttgtttaa ctttaagaag gagatatatc catgaatgga ctgaatgata
      181 tctttgaagc gcagaaaatt gaatggcatg aatccggatc tcatcaccat caccatcacc
      241 atcacactag tatgaaaatc gaagaaggta aactggtaat ctggattaac ggcgataaag
      301 gctataacgg tctcgctgaa gtcggtaaga aattcgagaa agataccgga attaaagtca
      361 ccgttgagca tccggataaa ctggaagaga aattcccaca ggttgcggca actggcgatg
      421 gccctgacat tatcttctgg gcacacgacc gctttggtgg ctacgctcaa tctggcctgt
      481 tggctgaaat caccccggac aaagcgttcc aggacaagct gtatccgttt acctgggatg
      541 ccgtacgtta caacggcaag ctgattgctt acccgatcgc tgttgaagcg ttatcgctga
      601 tttataacaa agatctgctg ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg
      661 ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact
      721 tcacctggcc gctgattgct gctgacgggg gttatgcgtt caagtatgaa aacggcaagt
      781 acgacattaa agacgtgggc gtggataacg ctggcgcgaa agcgggtctg accttcctgg
      841 ttgacctgat taaaaacaaa cacatgaatg cagacaccga ttactccatc gcagaagctg
      901 cctttaataa aggcgaaaca gcgatgacca tcaacggccc gtgggcatgg tccaacatcg
      961 acaccagcaa agtgaattat ggtgtaacgg tactgccgac cttcaagggt caaccatcca
     1021 aaccgttcgt tggcgtgctg agcgcaggta ttgacgccgc cagtccgaac aaagagctgg
     1081 caaaagagtt cctcgaaaac tatctgctga ctgatgaagg tctggaagcg gttaataaag
     1141 acaaaccgct gggtgccgta gcgctgaagt cttacgagga agagttggcg aaagatccac
     1201 gtattgccgc cacaatggaa aacgcccaga aaggtgaaat catgccgaac atcccgcaga
     1261 tgtccgcttt ctggtatgcc gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga
     1321 ctgtcgatga agccctgaaa gacgcgcaga ctaattcgag ctcgaccggt agtggcacca
     1381 gtgggtcgac actggaagtt ctgtttcagg gcccaggatc catggaattc gcggccgccc
     1441 taggctcgag ctaagcttga gtattctata gtgtcaccta aatcccagct tgatccggct
     1501 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     1561 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     1621 tccggataac ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     1681 gcagcctgaa tggcgaatgg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     1741 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     1801 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     1861 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     1921 acagcttgca tgcctgcagg tcggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     1981 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     2041 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     2101 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     2161 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     2221 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     2281 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     2341 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     2401 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     2461 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     2521 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     2581 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     2641 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     2701 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     2761 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     2821 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     2881 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     2941 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     3001 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     3061 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     3121 ggcgctatca tgccataccg cgaaaggttt tgcaccattc gatggtgtcg ggtaccgagc
     3181 tcgaattgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     3241 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     3301 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     3361 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     3421 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc
     3481 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     3541 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     3601 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     3661 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     3721 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     3781 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     3841 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     3901 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     3961 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     4021 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     4081 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     4141 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     4201 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     4261 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     4321 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     4381 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     4441 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     4501 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     4561 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     4621 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     4681 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     4741 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     4801 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     4861 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     4921 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     4981 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     5041 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5101 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5161 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5221 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5281 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     5341 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     5401 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     5461 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     5521 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     5581 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtg
//
