LOCUS       sequence-220215-        5633 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 5633)
  TITLE     Hyvonen Lab pOP plasmids
REFERENCE   2  (bases 1 to 5633)
  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..5633
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     59..78
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        59..77
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    77..101
                     /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             114..136
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             150..194
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             204..227
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             234..1334
                     /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     1308..1331
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1374..1394
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        complement(1441..1459)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1442..1459)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1517..1535)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1531..1578
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1814..1835)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1985..3067)
                     /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     3029..3048
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3068..3145)
                     /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     3288..3307
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3461..3480)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3580..3602
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3640..3658)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3726..3830
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3831..3899,3900..4691)
                     /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(4049..4068)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4862..5450
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5351..5370
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5604..5621
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctgatta
       61 atacgactca ctatagggaa ttgtgagcgg ataacaattc ctctagaaat aattttgttt
      121 aactttaaga aggagatata tccatgaatg gactgaatga tatctttgaa gcgcagaaaa
      181 ttgaatggca tgaatccgga tctcatcacc atcaccatca ccatcacact agtatgaaaa
      241 tcgaagaagg taaactggta atctggatta acggcgataa aggctataac ggtctcgctg
      301 aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata
      361 aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac attatcttct
      421 gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg
      481 acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca
      541 agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc
      601 tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga
      661 aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg
      721 ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg
      781 gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca
      841 aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa
      901 cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt
      961 atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc
     1021 tgagcgcagg tattgacgcc gccagtccga acaaagagct ggcaaaagag ttcctcgaaa
     1081 actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg
     1141 tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc gccacaatgg
     1201 aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg
     1261 ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga
     1321 aagacgcgca gactaattcg agctcgaccg gtagtggcac cagtgggtcg acagaaaacc
     1381 tgtacttcca gggatccatg gaattcgcgg ccgccctagg ctcgagctaa gcttgagtat
     1441 tctatagtgt cacctaaatc ccagcttgat ccggctgcta acaaagcccg aaaggaagct
     1501 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     1561 gtcttgaggg gttttttgct gaaaggagga actatatccg gataacctgg cgtaatagcg
     1621 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggaaat
     1681 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     1741 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     1801 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     1861 caaagggcga aaaaccgtct atcagggcga tggcccacag cttgcatgcc tgcaggtcgg
     1921 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taacttacat taattgcgtt
     1981 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     2041 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     2101 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     2161 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     2221 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     2281 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     2341 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     2401 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     2461 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     2521 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     2581 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     2641 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     2701 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     2761 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     2821 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     2881 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     2941 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     3001 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     3061 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     3121 aggttttgca ccattcgatg gtgtcgggta ccgagctcga attgtgaacc atcaccctaa
     3181 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     3241 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     3301 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     3361 cccgccgcgc ttaatgcgcc gctacagggc gcgtcctgat gcggtatttt ctccttacgc
     3421 atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg
     3481 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     3541 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     3601 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     3661 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     3721 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3781 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     3841 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     3901 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     3961 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     4021 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     4081 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     4141 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     4201 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     4261 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     4321 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     4381 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     4441 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     4501 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     4561 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     4621 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     4681 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     4741 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4801 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4861 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4921 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4981 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     5041 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5101 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5161 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5221 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5281 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5341 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5401 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5461 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     5521 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     5581 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gag
//
