LOCUS       sequence-220234-        4847 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 4847)
  TITLE     Hyvonen Lab pOP plasmids
REFERENCE   2  (bases 1 to 4847)
  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..4847
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     46..65
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        46..64
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    64..88
                     /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             101..123
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             137..181
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             191..214
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             221..547
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             575..595
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        complement(642..660)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(643..660)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(718..736)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      732..779
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1015..1036)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1186..2268)
                     /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     2230..2249
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2269..2346)
                     /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     2489..2508
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2662..2681)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2781..2803
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2841..2859)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2927..3031
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3032..3100,3101..3892)
                     /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(3250..3269)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4063..4651
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4552..4571
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4805..4822
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctgattaata cgactcacta
       61 tagggaattg tgagcggata acaattcctc tagaaataat tttgtttaac tttaagaagg
      121 agatatatcc atgaatggac tgaatgatat ctttgaagcg cagaaaattg aatggcatga
      181 atccggatct catcaccatc accatcacca tcacactagt atgagcgata aaattattca
      241 cctgactgac gacagttttg acacggatgt actcaaagcg gacggggcga tcctcgtcga
      301 tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc ccgattctgg atgaaatcgc
      361 tgacgaatat cagggcaaac tgaccgttgc aaaactgaac atcgatcaaa accctggcac
      421 tgcgccgaaa tatggcatcc gtggtatccc gactctgctg ctgttcaaaa acggtgaagt
      481 ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg aaagagttcc tcgacgctaa
      541 cctggcgacc ggtagtggca ccagtgggtc gacagaaaac ctgtacttcc agggatccat
      601 ggaattcgcg gccgccctag gctcgagcta agcttgagta ttctatagtg tcacctaaat
      661 cccagcttga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
      721 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
      781 tgaaaggagg aactatatcc ggataacctg gcgtaatagc gaagaggccc gcaccgatcg
      841 cccttcccaa cagttgcgca gcctgaatgg cgaatggaaa ttgtaagcgt taatattttg
      901 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
      961 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     1021 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     1081 tatcagggcg atggcccaca gcttgcatgc ctgcaggtcg gaagcataaa gtgtaaagcc
     1141 tggggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     1201 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     1261 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     1321 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     1381 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     1441 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     1501 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     1561 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     1621 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     1681 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     1741 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     1801 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     1861 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     1921 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     1981 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     2041 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     2101 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     2161 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     2221 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     2281 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc accattcgat
     2341 ggtgtcgggt accgagctcg aattgtgaac catcacccta atcaagtttt ttggggtcga
     2401 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     2461 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     2521 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     2581 cgctacaggg cgcgtcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     2641 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     2701 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     2761 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     2821 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     2881 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     2941 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3001 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     3061 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     3121 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     3181 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     3241 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     3301 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     3361 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     3421 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     3481 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     3541 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     3601 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     3661 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     3721 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     3781 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     3841 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     3901 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     3961 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     4021 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4081 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4141 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     4201 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4261 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     4321 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     4381 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     4441 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     4501 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     4561 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     4621 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     4681 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     4741 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     4801 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgca
//
