LOCUS       sequence-212068-        4852 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 thioredoxin fusions.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4852)
  TITLE     Hyvonen Lab pOP plasmids
REFERENCE   2  (bases 1 to 4852)
  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: pOP3TT
FEATURES             Location/Qualifiers
     source          1..4852
                     /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..502
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             530..550
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        complement(597..615)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(598..615)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(673..691)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      687..734
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(970..991)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1141..2223)
                     /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     2185..2204
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2224..2301)
                     /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     2447..2466
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2622..2641)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2741..2763
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2801..2819)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2887..2991
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2992..3060,3061..3852)
                     /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      4023..4611
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4512..4531
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4765..4782
                     /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 gggtcgacag aaaacctgta
      541 cttccaggga tccatggaat tcgcggccgc cctaggctcg agctaagctt gagtattcta
      601 tagtgtcacc taaatcccag cttgatccgg ctgctaacaa agcccgaaag gaagctgagt
      661 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
      721 tgaggggttt tttgctgaaa ggaggaacta tatccggata acctggcgta atagcgaaga
      781 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggaaattgta
      841 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
      901 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
      961 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     1021 gggcgaaaaa ccgtctatca gggcgatggc ccacagcttg catgcctgca ggtcggaagc
     1081 ataaagtgta aagcctgggg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     1141 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     1201 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     1261 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     1321 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttgacg gcgggatata
     1381 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
     1441 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     1501 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     1561 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     1621 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     1681 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     1741 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     1801 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     1861 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     1921 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     1981 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     2041 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     2101 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     2161 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     2221 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     2281 tttgcaccat tcgatggtgt cgggtaccga gctcgaattt acgtgaacca tcacccaaat
     2341 caagtttttt gcggtcgagg tgccgtaaag ctctaaatcg gaaccctaaa gggagccccc
     2401 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     2461 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     2521 ccgccgcgct taatgcgccg ctacagggcg cgtcgcctga tgcggtattt tctccttacg
     2581 catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc
     2641 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     2701 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     2761 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     2821 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     2881 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     2941 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     3001 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     3061 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     3121 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     3181 tttccaatga tgagcacttt taaagttctg ctatgtcata cactattatc ccgtattgac
     3241 gccgggcaag agcaactcgg tcgccgggcg cggtattctc agaatgactt ggttgagtac
     3301 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     3361 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     3421 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     3481 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     3541 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     3601 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     3661 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     3721 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     3781 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     3841 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     3901 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     3961 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4021 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4081 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4141 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     4201 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4261 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4321 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4381 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4441 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4501 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4561 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4621 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4681 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     4741 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     4801 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg at
//
