LOCUS       sequence-212065-        4696 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 GB1 fusions.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4696)
  TITLE     Hyvonen Lab pOP plasmids
REFERENCE   2  (bases 1 to 4696)
  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: pOP3BP
FEATURES             Location/Qualifiers
     source          1..4696
                     /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             179..340
                     /codon_start=1
                     /product="B1 domain of Streptococcal protein G   "
                     /label=GB1
                     /note="effective as a solubilizing fusion partner (Cheng 
                     and Patel, 2004)"
                     /translation="YKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDD
                     ATKTFTVTE"
     CDS             368..391
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        complement(441..459)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(442..459)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(517..535)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      531..578
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(814..835)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(985..2067)
                     /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     2029..2048
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2068..2145)
                     /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     2291..2310
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2466..2485)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2585..2607
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2645..2663)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2731..2835
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2836..2904,2905..3696)
                     /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      3867..4455
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4356..4375
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4609..4626
                     /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 ctagtaccta
      181 caaactgatc ctgaacggta aaaccctgaa aggtgaaacc accaccgaag ctgtagacgc
      241 tgctactgct gaaaaagttt tcaaacagta cgctaacgac aacggtgtgg acggtgaatg
      301 gacctacgac gacgctacca aaaccttcac ggttacggaa accggtagtg gcaccagtgg
      361 gtcgacactg gaagttctgt ttcagggccc aggatccatg gaattcgcgg ccgccctagg
      421 ctcgagctaa gcttgagtat tctatagtgt cacctaaatc ccagcttgat ccggctgcta
      481 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
      541 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
      601 gataacctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
      661 cctgaatggc gaatggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
      721 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
      781 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
      841 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacag
      901 cttgcatgcc tgcaggtcgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
      961 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     1021 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     1081 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     1141 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     1201 gatggtggtt gacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     1261 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     1321 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     1381 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     1441 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     1501 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     1561 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     1621 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     1681 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     1741 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     1801 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     1861 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     1921 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     1981 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     2041 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     2101 ctatcatgcc ataccgcgaa aggttttgca ccattcgatg gtgtcgggta ccgagctcga
     2161 atttacgtga accatcaccc aaatcaagtt ttttgcggtc gaggtgccgt aaagctctaa
     2221 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     2281 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     2341 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcgc
     2401 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact
     2461 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     2521 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     2581 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     2641 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     2701 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2761 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2821 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     2881 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     2941 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     3001 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     3061 catacactat tatcccgtat tgacgccggg caagagcaac tcggtcgccg ggcgcggtat
     3121 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     3181 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     3241 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     3301 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     3361 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     3421 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     3481 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     3541 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     3601 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3661 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     3721 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     3781 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3841 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3901 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3961 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     4021 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4081 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4141 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4201 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4261 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4321 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4381 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4441 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4501 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4561 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4621 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     4681 cattaatgca gctgat
//
