LOCUS       sequence-220216-        4691 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Used for expression of protein in E. coli as PreScission 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 4691)
  TITLE     Hyvonen Lab pOP plasmids
REFERENCE   2  (bases 1 to 4691)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pOP3BP
FEATURES             Location/Qualifiers
     source          1..4691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     3..20
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     91..110
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        91..109
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    109..133
                     /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             146..168
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             182..226
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             236..259
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             269..430
                     /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             458..481
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        complement(531..549)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(532..549)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(607..625)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      621..668
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(904..925)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1075..2157)
                     /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     2119..2138
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2158..2235)
                     /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     2378..2397
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2551..2570)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2670..2692
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2730..2748)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2816..2920
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2921..2989,2990..3781)
                     /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(3139..3158)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3952..4540
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4441..4460
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
       61 cgcgttggcc gattcattaa tgcagctgat taatacgact cactataggg aattgtgagc
      121 ggataacaat tcctctagaa ataattttgt ttaactttaa gaaggagata tatccatgaa
      181 tggactgaat gatatctttg aagcgcagaa aattgaatgg catgaatccg gatctcatca
      241 ccatcaccat caccatcaca ctagtaccta caaactgatc ctgaacggta aaaccctgaa
      301 aggtgaaacc accaccgaag ctgtagacgc tgctactgct gaaaaagttt tcaaacagta
      361 cgctaacgac aacggtgtgg acggtgaatg gacctacgac gacgctacca aaaccttcac
      421 ggttacggaa accggtagtg gcaccagtgg gtcgacactg gaagttctgt ttcagggccc
      481 aggatccatg gaattcgcgg ccgccctagg ctcgagctaa gcttgagtat tctatagtgt
      541 cacctaaatc ccagcttgat ccggctgcta acaaagcccg aaaggaagct gagttggctg
      601 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
      661 gttttttgct gaaaggagga actatatccg gataacctgg cgtaatagcg aagaggcccg
      721 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggaaat tgtaagcgtt
      781 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
      841 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
      901 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
      961 aaaaccgtct atcagggcga tggcccacag cttgcatgcc tgcaggtcgg aagcataaag
     1021 tgtaaagcct ggggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     1081 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     1141 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     1201 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     1261 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     1321 gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga
     1381 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     1441 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     1501 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     1561 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     1621 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     1681 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     1741 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     1801 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     1861 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     1921 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     1981 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     2041 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     2101 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     2161 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgca
     2221 ccattcgatg gtgtcgggta ccgagctcga attgtgaacc atcaccctaa tcaagttttt
     2281 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     2341 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     2401 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     2461 ttaatgcgcc gctacagggc gcgtcctgat gcggtatttt ctccttacgc atctgtgcgg
     2521 tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     2581 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     2641 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     2701 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     2761 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     2821 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     2881 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     2941 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     3001 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     3061 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     3121 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     3181 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     3241 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     3301 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     3361 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     3421 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     3481 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     3541 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     3601 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     3661 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     3721 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     3781 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     3841 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     3901 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     3961 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     4021 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     4081 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4141 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4201 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4261 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4321 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     4381 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     4441 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     4501 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     4561 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     4621 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     4681 aacgaccgag c
//
