LOCUS       sequence-220237-        4691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as PreScission 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 4691)
  TITLE     Hyvonen Lab pOP plasmids
REFERENCE   2  (bases 1 to 4691)
  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: pOP5BP
FEATURES             Location/Qualifiers
     source          1..4691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     39..58
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        39..57
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    57..81
                     /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             94..116
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             136..159
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             169..330
                     /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             358..381
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             418..462
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     promoter        complement(479..497)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(480..497)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(555..573)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      569..616
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(852..873)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1023..2105)
                     /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     2067..2086
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2106..2183)
                     /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     2326..2345
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2499..2518)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2618..2640
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2678..2696)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2764..2868
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2869..2937,2938..3729)
                     /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(3087..3106)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3900..4488
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4389..4408
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4642..4659
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tctccccgcg cgttggccga ttcattaatg cagctgatta atacgactca ctatagggaa
       61 ttgtgagcgg ataacaattc ctctagaaat aattttgttt aactttaaga aggagatata
      121 tccatgaatg gatctcatca ccatcaccat caccatcaca ctagtaccta caaactgatc
      181 ctgaacggta aaaccctgaa aggtgaaacc accaccgaag ctgtagacgc tgctactgct
      241 gaaaaagttt tcaaacagta cgctaacgac aacggtgtgg acggtgaatg gacctacgac
      301 gacgctacca aaaccttcac ggttacggaa accggtagtg gcaccagtgg gtcgacactg
      361 gaagttctgt ttcagggccc aggatccatg gaattcgcgg ccgccctagg ctcgagcgga
      421 ctgaatgaca ttttcgaagc acagaagatc gaatggcatg aagcctaagc ttgagtattc
      481 tatagtgtca cctaaatccc agcttgatcc ggctgctaac aaagcccgaa aggaagctga
      541 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
      601 cttgaggggt tttttgctga aaggaggaac tatatccgga taacctggcg taatagcgaa
      661 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggaaattg
      721 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
      781 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
      841 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
      901 aagggcgaaa aaccgtctat cagggcgatg gcccacagct tgcatgcctg caggtcggaa
      961 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta acttacatta attgcgttgc
     1021 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     1081 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     1141 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     1201 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     1261 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     1321 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     1381 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     1441 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     1501 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     1561 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     1621 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     1681 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     1741 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     1801 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     1861 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     1921 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     1981 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     2041 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     2101 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     2161 gttttgcacc attcgatggt gtcgggtacc gagctcgaat tgtgaaccat caccctaatc
     2221 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     2281 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     2341 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     2401 cgccgcgctt aatgcgccgc tacagggcgc gtcctgatgc ggtattttct ccttacgcat
     2461 ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca
     2521 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     2581 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     2641 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     2701 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     2761 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2821 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     2881 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     2941 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     3001 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     3061 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     3121 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     3181 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     3241 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     3301 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     3361 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     3421 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     3481 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     3541 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     3601 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     3661 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     3721 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     3781 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     3841 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3901 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3961 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4021 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     4081 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4141 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4201 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4261 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4321 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4381 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4441 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4501 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4561 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4621 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     4681 cgcaaaccgc c
//
