LOCUS       sequence-220247-        5180 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as TEV cleavable 
            N-terminual 8His and GST tag fusions and with C-terminal Avi-tag .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5180)
  TITLE     Hyvonen Lab pOP plasmids
REFERENCE   2  (bases 1 to 5180)
  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: pOP5GT
FEATURES             Location/Qualifiers
     source          1..5180
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     31..50
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        31..49
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    49..73
                     /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             86..108
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             128..151
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             167..817
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPP"
     primer_bind     778..800
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             845..865
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             899..943
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     promoter        complement(960..978)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(961..978)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1036..1054)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1050..1097
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1333..1354)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1504..2586)
                     /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     2548..2567
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2587..2664)
                     /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     2807..2826
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2980..2999)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3099..3121
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3159..3177)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3245..3349
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3350..3418,3419..4210)
                     /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(3568..3587)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4381..4969
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4870..4889
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5123..5140
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgcgttggcc gattcattaa tgcagctgat taatacgact cactataggg aattgtgagc
       61 ggataacaat tcctctagaa ataattttgt ttaactttaa gaaggagata tatccatgaa
      121 tggatctcat caccatcacc atcaccatca cactagtagc aattccatgt cccctatact
      181 aggttattgg aaaattaagg gccttgtgca acccactcga cttcttttgg aatatcttga
      241 agaaaaatat gaagagcatt tgtatgagcg cgatgaaggt gataaatggc gaaacaaaaa
      301 gtttgaattg ggtttggagt ttcccaatct tccttattat attgatggtg atgttaaatt
      361 aacacagtct atggccatca tacgttatat agctgacaag cacaacatgt tgggtggttg
      421 tccaaaagag cgtgcagaga tttcaatgct tgaaggagcg gttttggata ttagatacgg
      481 tgtttcgaga attgcatata gtaaagactt tgaaactctc aaagttgatt ttcttagcaa
      541 gctacctgaa atgctgaaaa tgttcgaaga tcgtttatgt cataaaacat atttaaatgg
      601 tgatcatgta acccatcctg acttcatgtt gtatgacgct cttgatgttg ttttatacat
      661 ggacccaatg tgcctggatg cgttcccaaa attagtttgt tttaaaaaac gtattgaagc
      721 tatcccacaa attgataagt acttgaaatc cagcaagtat atagcatggc ctttgcaggg
      781 ctggcaagcc acgtttggtg gtggcgacca tcctccaacc ggtagtggca ccagtgggtc
      841 gacagaaaac ctgtacttcc agggatccat ggaattcgcg gccgccctag gctcgagcgg
      901 actgaatgac attttcgaag cacagaagat cgaatggcat gaagcctaag cttgagtatt
      961 ctatagtgtc acctaaatcc cagcttgatc cggctgctaa caaagcccga aaggaagctg
     1021 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     1081 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg ataacctggc gtaatagcga
     1141 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggaaatt
     1201 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     1261 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     1321 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     1381 aaagggcgaa aaaccgtcta tcagggcgat ggcccacagc ttgcatgcct gcaggtcgga
     1441 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aacttacatt aattgcgttg
     1501 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     1561 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     1621 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     1681 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     1741 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     1801 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     1861 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     1921 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     1981 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     2041 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     2101 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     2161 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     2221 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     2281 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     2341 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     2401 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     2461 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     2521 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     2581 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     2641 ggttttgcac cattcgatgg tgtcgggtac cgagctcgaa ttgtgaacca tcaccctaat
     2701 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     2761 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     2821 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     2881 ccgccgcgct taatgcgccg ctacagggcg cgtcctgatg cggtattttc tccttacgca
     2941 tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc
     3001 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     3061 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     3121 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     3181 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     3241 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     3301 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     3361 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     3421 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     3481 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     3541 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     3601 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     3661 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     3721 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     3781 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     3841 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     3901 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     3961 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     4021 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     4081 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     4141 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     4201 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     4261 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     4321 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     4381 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     4441 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4501 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     4561 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4621 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4681 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4741 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4801 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4861 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4921 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4981 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     5041 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     5101 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
     5161 acgcaaaccg cctctccccg
//
