LOCUS       sequence-220251-        4847 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as TEV cleavable 
            N-terminual 8His and thioredoxin tag fusions and with C-terminal 
            Avi-tag .
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: pOP5TT
FEATURES             Location/Qualifiers
     source          1..4847
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     44..63
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        44..62
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    62..86
                     /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             99..121
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             141..164
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             171..497
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             525..545
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             579..623
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     promoter        complement(640..658)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(641..658)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(716..734)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      730..777
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1013..1034)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1184..2266)
                     /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     2228..2247
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2267..2344)
                     /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     2487..2506
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2660..2679)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2779..2801
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2839..2857)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2925..3029
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3030..3098,3099..3890)
                     /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(3248..3267)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4061..4649
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4550..4569
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4803..4820
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct gattaatacg actcactata
       61 gggaattgtg agcggataac aattcctcta gaaataattt tgtttaactt taagaaggag
      121 atatatccat gaatggatct catcaccatc accatcacca tcacactagt atgagcgata
      181 aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg gacggggcga
      241 tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc ccgattctgg
      301 atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac atcgatcaaa
      361 accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg ctgttcaaaa
      421 acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg aaagagttcc
      481 tcgacgctaa cctggcgacc ggtagtggca ccagtgggtc gacagaaaac ctgtacttcc
      541 agggatccat ggaattcgcg gccgccctag gctcgagcgg actgaatgac attttcgaag
      601 cacagaagat cgaatggcat gaagcctaag cttgagtatt ctatagtgtc acctaaatcc
      661 cagcttgatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
      721 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
      781 aaaggaggaa ctatatccgg ataacctggc gtaatagcga agaggcccgc accgatcgcc
      841 cttcccaaca gttgcgcagc ctgaatggcg aatggaaatt gtaagcgtta atattttgtt
      901 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
      961 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     1021 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     1081 tcagggcgat ggcccacagc ttgcatgcct gcaggtcgga agcataaagt gtaaagcctg
     1141 gggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     1201 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     1261 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     1321 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     1381 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     1441 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     1501 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     1561 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     1621 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     1681 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     1741 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     1801 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     1861 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     1921 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     1981 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     2041 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     2101 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     2161 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     2221 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     2281 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcac cattcgatgg
     2341 tgtcgggtac cgagctcgaa ttgtgaacca tcaccctaat caagtttttt ggggtcgagg
     2401 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     2461 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     2521 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     2581 ctacagggcg cgtcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     2641 gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     2701 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     2761 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     2821 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     2881 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     2941 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     3001 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     3061 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     3121 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     3181 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     3241 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     3301 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     3361 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     3421 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     3481 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     3541 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     3601 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     3661 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     3721 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     3781 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     3841 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     3901 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     3961 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     4021 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     4081 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4141 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4201 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4261 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4321 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4381 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4441 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4501 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4561 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4621 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4681 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4741 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     4801 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaa
//
