LOCUS       sequence-217222-        4955 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as TEV cleavable 
            N-terminual 8His and PpiB (peptidyl-prolyl cis-trans isomerase B 
            from E. coli) fusions with optional C-terminal StrepII-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4955)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 4955)
  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"; journalName: 
            "unpublished"
COMMENT     Sequence Label: pExp-PpiB
FEATURES             Location/Qualifiers
     source          1..4955
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    19..43
                     /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             56..78
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..115
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             672..695
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(709..727)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(710..727)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(785..803)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      799..846
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1082..1103)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1253..2335)
                     /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     2297..2316
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2336..2413)
                     /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     2552..2571
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2725..2744)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2844..2866
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2904..2922)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2990..3094
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3095..3163,3164..3955)
                     /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(3313..3332)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4126..4714
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4615..4634
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4868..4885
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tcaccatcac catcaccatc accattctgg
      121 cactagtggc gttactttcc acaccaatca cggcgatatt gtcatcaaaa cttttgacga
      181 taaagcacct gaaacagtta aaaacttcct ggactactgc cgcgaaggtt tttacaacaa
      241 caccattttc caccgtgtta tcaacggctt tatgattcag ggcggcggtt ttgaaccggg
      301 catgaaacaa aaagccacca aagaaccgat caaaaacgaa gccaacaacg gcctgaaaaa
      361 tacccgtggt acgctggcaa tggcacgtac tcaggctccg cactctgcaa ctgcacagtt
      421 cttcatcaac gtggttgata acgacttcct gaacttctct ggcgaaagcc tgcaaggttg
      481 gggctactgc gtgtttgctg aagtggttga cggcatggac gtggtagaca aaatcaaagg
      541 tgttgcaacc ggtcgtagcg gtatgcacca ggacgtgcca aaagaagacg ttatcattga
      601 aagcgtgacc gttagcgaga gcggtaccga aaacctgtac ttccagtgag accttaatta
      661 actcgagcgc atggagccac ccgcagttcg aaaaataagc ttgagtattc tatagtgtca
      721 cctaaatccc agcttgatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
      781 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
      841 tttttgctga aaggaggaac tatatccgga taacctggcg taatagcgaa gaggcccgca
      901 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggaaattg taagcgttaa
      961 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     1021 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     1081 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     1141 aaccgtctat cagggcgatg gcccacagct tgcatgcctg caggtcggaa gcataaagtg
     1201 taaagcctgg ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     1261 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     1321 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     1381 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     1441 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     1501 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     1561 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     1621 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     1681 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     1741 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     1801 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     1861 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     1921 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     1981 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     2041 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     2101 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     2161 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     2221 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     2281 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     2341 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcacc
     2401 attcgatggt gtcggcgagc tcgaattgtg aaccatcacc ctaatcaagt tttttggggt
     2461 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     2521 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     2581 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     2641 cgccgctaca gggcgcgtcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     2701 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     2761 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     2821 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     2881 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     2941 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     3001 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     3061 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     3121 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     3181 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     3241 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     3301 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     3361 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     3421 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     3481 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     3541 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     3601 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     3661 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     3721 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     3781 tgctgataaa tctggagccg gtgagcgtgg ttctcgcggt atcattgcag cactggggcc
     3841 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     3901 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     3961 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     4021 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     4081 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     4141 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     4201 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     4261 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     4321 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     4381 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     4441 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     4501 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     4561 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     4621 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     4681 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     4741 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     4801 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     4861 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     4921 ccccgcgcgt tggccgattc attaatgcag ctgat
//
