LOCUS       sequence-220219-        4853 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 thioredoxin fusions.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4853)
  TITLE     Hyvonen Lab pOP plasmids
REFERENCE   2  (bases 1 to 4853)
  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: pOP3TP
FEATURES             Location/Qualifiers
     source          1..4853
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     74..93
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        74..92
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    92..116
                     /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             129..151
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             165..209
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             219..242
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             249..575
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             603..626
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        complement(676..694)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(677..694)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(752..770)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      766..813
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1049..1070)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1220..2302)
                     /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     2264..2283
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2303..2380)
                     /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     2523..2542
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2696..2715)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2815..2837
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2875..2893)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2961..3065
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3066..3134,3135..3926)
                     /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(3284..3303)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4097..4685
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4586..4605
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(4839..4853,1..3)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
       61 taatgcagct gattaatacg actcactata gggaattgtg agcggataac aattcctcta
      121 gaaataattt tgtttaactt taagaaggag atatatccat gaatggactg aatgatatct
      181 ttgaagcgca gaaaattgaa tggcatgaat ccggatctca tcaccatcac catcaccatc
      241 acactagtat gagcgataaa attattcacc tgactgacga cagttttgac acggatgtac
      301 tcaaagcgga cggggcgatc ctcgtcgatt tctgggcaga gtggtgcggt ccgtgcaaaa
      361 tgatcgcccc gattctggat gaaatcgctg acgaatatca gggcaaactg accgttgcaa
      421 aactgaacat cgatcaaaac cctggcactg cgccgaaata tggcatccgt ggtatcccga
      481 ctctgctgct gttcaaaaac ggtgaagtgg cggcaaccaa agtgggtgca ctgtctaaag
      541 gtcagttgaa agagttcctc gacgctaacc tggcgaccgg tagtggcacc agtgggtcga
      601 cactggaagt tctgtttcag ggcccaggat ccatggaatt cgcggccgcc ctaggctcga
      661 gctaagcttg agtattctat agtgtcacct aaatcccagc ttgatccggc tgctaacaaa
      721 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
      781 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggataa
      841 cctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
      901 atggcgaatg gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
      961 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     1021 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     1081 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cacagcttgc
     1141 atgcctgcag gtcggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     1201 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     1261 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     1321 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     1381 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     1441 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     1501 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     1561 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     1621 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     1681 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     1741 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     1801 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     1861 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     1921 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     1981 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     2041 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     2101 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     2161 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     2221 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     2281 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     2341 atgccatacc gcgaaaggtt ttgcaccatt cgatggtgtc gggtaccgag ctcgaattgt
     2401 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     2461 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     2521 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     2581 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ctgatgcggt
     2641 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa
     2701 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     2761 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     2821 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     2881 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     2941 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     3001 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     3061 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     3121 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     3181 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     3241 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     3301 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     3361 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     3421 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     3481 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     3541 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     3601 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     3661 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     3721 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     3781 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     3841 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     3901 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     3961 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     4021 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     4081 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     4141 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     4201 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     4261 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     4321 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     4381 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     4441 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     4501 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     4561 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     4621 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     4681 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     4741 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     4801 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agc
//
