LOCUS       Exported                4858 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Used for expression of protein in E. coli as PreScission cleavable 
            N-terminal Avi-His8-tag and thioredoxin fusions.
ACCESSION   .
VERSION     .
KEYWORDS    pOP3TP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4858)
  TITLE     Hyvonen Lab pOP plasmids
REFERENCE   2  (bases 1 to 4858)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4858
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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..499
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANL"
     CDS             530..553
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        complement(603..621)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(604..621)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(679..697)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      693..740
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(976..997)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1147..2229)
                     /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     2191..2210
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2230..2307)
                     /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     2453..2472
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2628..2647)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2747..2769
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2807..2825)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2893..2997
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4029..4617
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4518..4537
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4771..4788
                     /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 ctagtatgag
      181 cgataaaatt attcacctga ctgacgacag ttttgacacg gatgtactca aagcggacgg
      241 ggcgatcctc gtcgatttct gggcagagtg gtgcggtccg tgcaaaatga tcgccccgat
      301 tctggatgaa atcgctgacg aatatcaggg caaactgacc gttgcaaaac tgaacatcga
      361 tcaaaaccct ggcactgcgc cgaaatatgg catccgtggt atcccgactc tgctgctgtt
      421 caaaaacggt gaagtggcgg caaccaaagt gggtgcactg tctaaaggtc agttgaaaga
      481 gttcctcgac gctaacctgg cgaccggtag tggcaccagt gggtcgacac tggaagttct
      541 gtttcagggc ccaggatcca tggaattcgc ggccgcccta ggctcgagct aagcttgagt
      601 attctatagt gtcacctaaa tcccagcttg atccggctgc taacaaagcc cgaaaggaag
      661 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
      721 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggataacct ggcgtaatag
      781 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggaa
      841 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
      901 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
      961 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     1021 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac agcttgcatg cctgcaggtc
     1081 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaacttac attaattgcg
     1141 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     1201 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     1261 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     1321 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttgacggcgg
     1381 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     1441 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     1501 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     1561 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     1621 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     1681 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     1741 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     1801 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     1861 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     1921 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     1981 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     2041 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     2101 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     2161 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     2221 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     2281 aaaggttttg caccattcga tggtgtcggg taccgagctc gaatttacgt gaaccatcac
     2341 ccaaatcaag ttttttgcgg tcgaggtgcc gtaaagctct aaatcggaac cctaaaggga
     2401 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     2461 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     2521 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcctgatgcg gtattttctc
     2581 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct
     2641 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     2701 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     2761 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     2821 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     2881 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2941 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     3001 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     3061 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     3121 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     3181 gaacgttttc caatgatgag cacttttaaa gttctgctat gtcatacact attatcccgt
     3241 attgacgccg ggcaagagca actcggtcgc cgggcgcggt attctcagaa tgacttggtt
     3301 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     3361 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     3421 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     3481 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     3541 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     3601 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     3661 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     3721 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     3781 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     3841 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     3901 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     3961 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4021 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4081 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4141 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     4201 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4261 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4321 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4381 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4441 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4501 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4561 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4621 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4681 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4741 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4801 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctgat
//
