LOCUS       sequence-220239-        4685 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as TEV cleavable 
            N-terminual 8His and GB1 tag fusions and with C-terminal Avi-tag .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4685)
  TITLE     Hyvonen Lab pOP plasmids
REFERENCE   2  (bases 1 to 4685)
  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: pOP5BT
FEATURES             Location/Qualifiers
     source          1..4685
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     49..68
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        49..67
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    67..91
                     /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             104..126
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             146..169
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             179..340
                     /codon_start=1
                     /product="B1 domain of Streptococcal protein G   "
                     /label=GB1
                     /note="effective as a solubilizing fusion partner (Cheng 
                     and Patel, 2004)"
                     /translation="YKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDD
                     ATKTFTVTE"
     CDS             368..388
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             422..466
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     promoter        complement(483..501)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(484..501)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(559..577)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      573..620
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(856..877)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1027..2109)
                     /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     2071..2090
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2110..2187)
                     /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     2330..2349
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2503..2522)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2622..2644
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2682..2700)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2768..2872
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2873..2941,2942..3733)
                     /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(3091..3110)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3904..4492
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4393..4412
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4646..4663
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctgatta atacgactca
       61 ctatagggaa ttgtgagcgg ataacaattc ctctagaaat aattttgttt aactttaaga
      121 aggagatata tccatgaatg gatctcatca ccatcaccat caccatcaca ctagtaccta
      181 caaactgatc ctgaacggta aaaccctgaa aggtgaaacc accaccgaag ctgtagacgc
      241 tgctactgct gaaaaagttt tcaaacagta cgctaacgac aacggtgtgg acggtgaatg
      301 gacctacgac gacgctacca aaaccttcac ggttacggaa accggtagtg gcaccagtgg
      361 gtcgacagaa aacctgtact tccagggatc catggaattc gcggccgccc taggctcgag
      421 cggactgaat gacattttcg aagcacagaa gatcgaatgg catgaagcct aagcttgagt
      481 attctatagt gtcacctaaa tcccagcttg atccggctgc taacaaagcc cgaaaggaag
      541 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
      601 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggataacct ggcgtaatag
      661 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggaa
      721 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
      781 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
      841 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
      901 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac agcttgcatg cctgcaggtc
      961 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaacttac attaattgcg
     1021 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     1081 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     1141 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     1201 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     1261 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     1321 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     1381 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     1441 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     1501 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     1561 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     1621 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     1681 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     1741 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     1801 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     1861 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     1921 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     1981 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     2041 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     2101 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     2161 aaaggttttg caccattcga tggtgtcggg taccgagctc gaattgtgaa ccatcaccct
     2221 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     2281 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     2341 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     2401 cacccgccgc gcttaatgcg ccgctacagg gcgcgtcctg atgcggtatt ttctccttac
     2461 gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc
     2521 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     2581 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     2641 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     2701 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     2761 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     2821 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     2881 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     2941 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     3001 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     3061 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     3121 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     3181 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     3241 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     3301 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     3361 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     3421 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     3481 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     3541 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     3601 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     3661 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     3721 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     3781 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     3841 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     3901 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     3961 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     4021 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     4081 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4141 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4201 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4261 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     4321 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     4381 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     4441 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     4501 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     4561 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     4621 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     4681 aatac
//
