LOCUS       sequence-220218-        4685 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 GB1 fusions.
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: pOP3BT
FEATURES             Location/Qualifiers
     source          1..4685
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..38
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     109..128
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        109..127
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    127..151
                     /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             164..186
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             200..244
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             254..277
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             287..448
                     /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             476..496
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        complement(543..561)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(544..561)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(619..637)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      633..680
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(916..937)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1087..2169)
                     /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     2131..2150
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2170..2247)
                     /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     2390..2409
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2563..2582)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2682..2704
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2742..2760)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2828..2932
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2933..3001,3002..3793)
                     /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(3151..3170)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3964..4552
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4453..4472
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
       61 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctgatta atacgactca
      121 ctatagggaa ttgtgagcgg ataacaattc ctctagaaat aattttgttt aactttaaga
      181 aggagatata tccatgaatg gactgaatga tatctttgaa gcgcagaaaa ttgaatggca
      241 tgaatccgga tctcatcacc atcaccatca ccatcacact agtacctaca aactgatcct
      301 gaacggtaaa accctgaaag gtgaaaccac caccgaagct gtagacgctg ctactgctga
      361 aaaagttttc aaacagtacg ctaacgacaa cggtgtggac ggtgaatgga cctacgacga
      421 cgctaccaaa accttcacgg ttacggaaac cggtagtggc accagtgggt cgacagaaaa
      481 cctgtacttc cagggatcca tggaattcgc ggccgcccta ggctcgagct aagcttgagt
      541 attctatagt gtcacctaaa tcccagcttg atccggctgc taacaaagcc cgaaaggaag
      601 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
      661 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggataacct ggcgtaatag
      721 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggaa
      781 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
      841 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
      901 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
      961 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac agcttgcatg cctgcaggtc
     1021 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaacttac attaattgcg
     1081 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     1141 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     1201 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     1261 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     1321 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     1381 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     1441 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     1501 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     1561 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     1621 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     1681 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     1741 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     1801 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     1861 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     1921 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     1981 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     2041 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     2101 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     2161 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     2221 aaaggttttg caccattcga tggtgtcggg taccgagctc gaattgtgaa ccatcaccct
     2281 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     2341 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     2401 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     2461 cacccgccgc gcttaatgcg ccgctacagg gcgcgtcctg atgcggtatt ttctccttac
     2521 gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc
     2581 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     2641 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     2701 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     2761 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     2821 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     2881 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     2941 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     3001 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     3061 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     3121 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     3181 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     3241 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     3301 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     3361 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     3421 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     3481 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     3541 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     3601 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     3661 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     3721 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     3781 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     3841 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     3901 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     3961 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     4021 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     4081 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     4141 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4201 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4261 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4321 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     4381 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     4441 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     4501 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     4561 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     4621 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     4681 tcgcc
//
