LOCUS       sequence-255699-        4835 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as TEV cleavable 
            N-terminual 8His and MsyB (acidic protein MsyB 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 4835)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 4835)
  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            "unpublished"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-MsyB
FEATURES             Location/Qualifiers
     source          1..4835
                     /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             552..575
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(589..607)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(590..607)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(665..683)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      679..726
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(962..983)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1133..2215)
                     /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     2177..2196
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2216..2293)
                     /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     2432..2451
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2605..2624)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2724..2746
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2784..2802)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2870..2974
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2975..3043,3044..3835)
                     /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(3193..3212)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4006..4594
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4495..4514
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4748..4765
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tcaccatcac catcaccatc accattctgg
      121 cactagtggc accatgtacg caacgcttga agaagccatt gacgctgcac gcgaagaatt
      181 tcttgcagac aaccccggca tcgacgccga agatgcgaat gtgcaacagt tcaatgccca
      241 aaaatacgtt ttgcaggacg gcgacatcat gtggcaagtt gagttttttg ccgacgaagg
      301 ggaagaaggt gaatgtttac ctatgcttag cggtgaagcc gcgcaaagtg tttttgatgg
      361 cgactatgat gagatagaga tacgccagga gtggcaggaa gagaatacat tacatgaatg
      421 ggacgagggg gaatttcagc ttgagccacc gctggatacc gaggaaggac gcgcagcagc
      481 tgatgagtgg gatgaacgta gcggtaccga aaacctgtac ttccagtgag accttaatta
      541 actcgagcgc atggagccac ccgcagttcg aaaaataagc ttgagtattc tatagtgtca
      601 cctaaatccc agcttgatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
      661 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
      721 tttttgctga aaggaggaac tatatccgga taacctggcg taatagcgaa gaggcccgca
      781 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggaaattg taagcgttaa
      841 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
      901 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
      961 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     1021 aaccgtctat cagggcgatg gcccacagct tgcatgcctg caggtcggaa gcataaagtg
     1081 taaagcctgg ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     1141 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     1201 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     1261 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     1321 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     1381 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     1441 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     1501 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     1561 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     1621 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     1681 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     1741 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     1801 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     1861 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     1921 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     1981 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     2041 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     2101 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     2161 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     2221 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcacc
     2281 attcgatggt gtcggcgagc tcgaattgtg aaccatcacc ctaatcaagt tttttggggt
     2341 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     2401 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     2461 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     2521 cgccgctaca gggcgcgtcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     2581 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     2641 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     2701 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     2761 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     2821 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     2881 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     2941 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     3001 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     3061 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     3121 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     3181 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     3241 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     3301 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     3361 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     3421 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     3481 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     3541 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     3601 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     3661 tgctgataaa tctggagccg gtgagcgtgg ttctcgcggt atcattgcag cactggggcc
     3721 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     3781 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     3841 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     3901 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     3961 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     4021 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     4081 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     4141 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     4201 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     4261 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     4321 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     4381 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     4441 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     4501 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     4561 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     4621 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     4681 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     4741 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     4801 ccccgcgcgt tggccgattc attaatgcag ctgat
//
