LOCUS       sequence-217221-        5033 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as TEV cleavable 
            N-terminual 8His and DsbA (leaderless thiol:disulfide interchange 
            protein DsbA 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 5033)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 5033)
  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            "unpublished"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-DsbA(SS)
FEATURES             Location/Qualifiers
     source          1..5033
                     /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             750..773
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(787..805)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(788..805)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(863..881)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      877..924
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1160..1181)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1331..2413)
                     /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     2375..2394
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2414..2491)
                     /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     2630..2649
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2803..2822)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2922..2944
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2982..3000)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3068..3172
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3173..3241,3242..4033)
                     /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(3391..3410)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4204..4792
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4693..4712
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4946..4963
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tcaccatcac catcaccatc accattctgg
      121 cactagtggc gcgcagtatg aagatggtaa acagtacact accctggaaa aaccggtagc
      181 tggcgcgccg caagtgctgg agtttttctc tttcttcagc ccgcacagct atcagtttga
      241 agaagttctg catatttctg ataatgtgaa gaaaaaactg ccggaaggcg tgaagatgac
      301 taaataccac gtcaacttca tgggtggtga cctgggcaaa gatctgactc aggcatgggc
      361 tgtggcgatg gcgctgggcg tggaagacaa agtgactgtt ccgctgtttg aaggcgtaca
      421 gaaaacccag accattcgtt ctgcttctga tatccgcgat gtatttatca acgcaggtat
      481 taaaggtgaa gagtacgacg cggcgtggaa cagcttcgtg gtgaaatctc tggtcgctca
      541 gcaggaaaaa gctgcagctg acgtgcaatt gcgtggcgtt ccggcgatgt ttgttaacgg
      601 taaatatcag ctgaatccgc agggtatgga taccagcaat atggatgttt ttgttcagca
      661 gtatgctgat acagtgaaat atctgtccga gaaaaaatct ggtaccgaaa acctgtactt
      721 ccagtgagac cttaattaac tcgagcgcat ggagccaccc gcagttcgaa aaataagctt
      781 gagtattcta tagtgtcacc taaatcccag cttgatccgg ctgctaacaa agcccgaaag
      841 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
      901 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggata acctggcgta
      961 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     1021 ggaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     1081 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     1141 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     1201 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccacagcttg catgcctgca
     1261 ggtcggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac ttacattaat
     1321 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     1381 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     1441 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     1501 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     1561 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     1621 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     1681 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     1741 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     1801 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     1861 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     1921 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     1981 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     2041 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     2101 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     2161 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     2221 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     2281 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     2341 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     2401 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     2461 cgcgaaaggt tttgcaccat tcgatggtgt cggcgagctc gaattgtgaa ccatcaccct
     2521 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     2581 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     2641 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     2701 cacccgccgc gcttaatgcg ccgctacagg gcgcgtcctg atgcggtatt ttctccttac
     2761 gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc
     2821 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     2881 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     2941 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     3001 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     3061 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3121 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     3181 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     3241 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     3301 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     3361 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     3421 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     3481 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     3541 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     3601 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     3661 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     3721 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     3781 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     3841 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggtt ctcgcggtat
     3901 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     3961 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     4021 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     4081 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     4141 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     4201 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     4261 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     4321 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     4381 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4441 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4501 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4561 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     4621 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     4681 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     4741 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     4801 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     4861 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     4921 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     4981 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct gat
//
