LOCUS       sequence-212064-        5638 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as TEV 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 5638)
  TITLE     Hyvonen Lab pOP plasmids
REFERENCE   2  (bases 1 to 5638)
  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: pOP3MT
FEATURES             Location/Qualifiers
     source          1..5638
                     /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             92..136
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             146..169
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             176..1276
                     /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     1250..1273
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1316..1336
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        complement(1383..1401)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1384..1401)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1459..1477)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1473..1520
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1756..1777)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1927..3009)
                     /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     2971..2990
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3010..3087)
                     /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     3233..3252
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3408..3427)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3527..3549
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3587..3605)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3673..3777
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3778..3846,3847..4638)
                     /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      4809..5397
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5298..5317
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5551..5568
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tggactgaat gatatctttg aagcgcagaa
      121 aattgaatgg catgaatccg gatctcatca ccatcaccat caccatcaca ctagtatgaa
      181 aatcgaagaa ggtaaactgg taatctggat taacggcgat aaaggctata acggtctcgc
      241 tgaagtcggt aagaaattcg agaaagatac cggaattaaa gtcaccgttg agcatccgga
      301 taaactggaa gagaaattcc cacaggttgc ggcaactggc gatggccctg acattatctt
      361 ctgggcacac gaccgctttg gtggctacgc tcaatctggc ctgttggctg aaatcacccc
      421 ggacaaagcg ttccaggaca agctgtatcc gtttacctgg gatgccgtac gttacaacgg
      481 caagctgatt gcttacccga tcgctgttga agcgttatcg ctgatttata acaaagatct
      541 gctgccgaac ccgccaaaaa cctgggaaga gatcccggcg ctggataaag aactgaaagc
      601 gaaaggtaag agcgcgctga tgttcaacct gcaagaaccg tacttcacct ggccgctgat
      661 tgctgctgac gggggttatg cgttcaagta tgaaaacggc aagtacgaca ttaaagacgt
      721 gggcgtggat aacgctggcg cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa
      781 caaacacatg aatgcagaca ccgattactc catcgcagaa gctgccttta ataaaggcga
      841 aacagcgatg accatcaacg gcccgtgggc atggtccaac atcgacacca gcaaagtgaa
      901 ttatggtgta acggtactgc cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt
      961 gctgagcgca ggtattgacg ccgccagtcc gaacaaagag ctggcaaaag agttcctcga
     1021 aaactatctg ctgactgatg aaggtctgga agcggttaat aaagacaaac cgctgggtgc
     1081 cgtagcgctg aagtcttacg aggaagagtt ggcgaaagat ccacgtattg ccgccacaat
     1141 ggaaaacgcc cagaaaggtg aaatcatgcc gaacatcccg cagatgtccg ctttctggta
     1201 tgccgtgcgt actgcggtga tcaacgccgc cagcggtcgt cagactgtcg atgaagccct
     1261 gaaagacgcg cagactaatt cgagctcgac cggtagtggc accagtgggt cgacagaaaa
     1321 cctgtacttc cagggatcca tggaattcgc ggccgcccta ggctcgagct aagcttgagt
     1381 attctatagt gtcacctaaa tcccagcttg atccggctgc taacaaagcc cgaaaggaag
     1441 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     1501 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggataacct ggcgtaatag
     1561 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggaa
     1621 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     1681 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     1741 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     1801 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac agcttgcatg cctgcaggtc
     1861 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaacttac attaattgcg
     1921 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     1981 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     2041 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     2101 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttgacggcgg
     2161 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     2221 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     2281 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     2341 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     2401 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     2461 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     2521 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     2581 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     2641 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     2701 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     2761 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     2821 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     2881 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     2941 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     3001 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     3061 aaaggttttg caccattcga tggtgtcggg taccgagctc gaatttacgt gaaccatcac
     3121 ccaaatcaag ttttttgcgg tcgaggtgcc gtaaagctct aaatcggaac cctaaaggga
     3181 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     3241 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     3301 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcctgatgcg gtattttctc
     3361 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct
     3421 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     3481 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     3541 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     3601 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     3661 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     3721 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     3781 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     3841 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     3901 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     3961 gaacgttttc caatgatgag cacttttaaa gttctgctat gtcatacact attatcccgt
     4021 attgacgccg ggcaagagca actcggtcgc cgggcgcggt attctcagaa tgacttggtt
     4081 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     4141 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     4201 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     4261 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     4321 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     4381 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     4441 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     4501 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     4561 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     4621 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     4681 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     4741 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4801 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4861 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4921 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     4981 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     5041 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5101 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5161 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5221 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5281 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5341 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     5401 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     5461 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     5521 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     5581 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctgat
//
