LOCUS       sequence-217224-        5567 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for expression of protein in E. coli as TEV cleavable 
            N-terminual 8His and MBP fusions with optional C-terminal 
            StrepII-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5567)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 5567)
  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            "unpublished"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-MBP
FEATURES             Location/Qualifiers
     source          1..5567
                     /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             131..1228
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGIDAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1202..1225
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1284..1307
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(1321..1339)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1322..1339)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1397..1415)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1411..1458
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1694..1715)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1865..2947)
                     /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     2909..2928
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2948..3025)
                     /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     3164..3183
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3337..3356)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3456..3478
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3516..3534)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3602..3706
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3707..3775,3776..4567)
                     /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(3925..3944)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4738..5326
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5227..5246
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5480..5497
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tcaccatcac catcaccatc accattctgg
      121 cactagtggc aaaatcgaag aaggtaaact ggtaatctgg attaacggcg ataaaggcta
      181 taacggcctc gctgaagtcg gtaagaaatt cgagaaagat accggaatta aagtcaccgt
      241 tgagcatccg gataaactgg aagagaaatt cccacaggtt gcggcaactg gcgatggccc
      301 tgacattatc ttctgggcac acgaccgctt tggtggctac gctcaatctg gcctgttggc
      361 tgaaatcacc ccggacaaag cgttccagga caagctgtat ccgtttacct gggatgccgt
      421 acgttacaac ggcaagctga ttgcttaccc gatcgctgtt gaagcgttat cgctgattta
      481 taacaaagat ctgctgccga acccgccaaa aacctgggaa gagatcccgg cgctggataa
      541 agaactgaaa gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac cgtacttcac
      601 ctggccgctg attgctgctg acgggggtta tgcgttcaag tatgaaaacg gcaagtacga
      661 cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct tcctggttga
      721 cctgattaaa aacaaacaca tgaatgcaga caccgattac tccatcgcag aagctgcctt
      781 taataaaggc gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca acatcgacac
      841 cagcaaagtg aattatggtg taacggtact gccgaccttc aagggtcaac catccaaacc
      901 gttcgttggc gtgctgagcg caggtattga cgccgccagt ccgaacaaag agctggcaaa
      961 agagttcctc gaaaactatc tgctgactga tgaaggtctg gaagcggtta ataaagacaa
     1021 accgctgggt gccgtagcgc tgaagtctta cgaggaagag ttggcgaaag atccacgtat
     1081 tgccgccaca atggaaaacg cccagaaagg tgaaatcatg ccgaacatcc cgcagatgtc
     1141 cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc gccagcggtc gtcagactgt
     1201 cgatgaagcc ctgaaagacg cgcagactaa tagcggtacc gaaaacctgt acttccagtg
     1261 agaccttaat taactcgagc gcatggagcc acccgcagtt cgaaaaataa gcttgagtat
     1321 tctatagtgt cacctaaatc ccagcttgat ccggctgcta acaaagcccg aaaggaagct
     1381 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     1441 gtcttgaggg gttttttgct gaaaggagga actatatccg gataacctgg cgtaatagcg
     1501 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggaaat
     1561 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     1621 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     1681 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     1741 caaagggcga aaaaccgtct atcagggcga tggcccacag cttgcatgcc tgcaggtcgg
     1801 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taacttacat taattgcgtt
     1861 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     1921 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     1981 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     2041 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     2101 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     2161 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     2221 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     2281 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     2341 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     2401 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     2461 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     2521 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     2581 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     2641 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     2701 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     2761 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     2821 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     2881 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     2941 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     3001 aggttttgca ccattcgatg gtgtcggcga gctcgaattg tgaaccatca ccctaatcaa
     3061 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     3121 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     3181 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     3241 ccgcgcttaa tgcgccgcta cagggcgcgt cctgatgcgg tattttctcc ttacgcatct
     3301 gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata
     3361 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     3421 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     3481 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     3541 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     3601 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     3661 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     3721 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     3781 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     3841 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     3901 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     3961 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     4021 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     4081 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     4141 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     4201 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     4261 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     4321 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     4381 tggctggttt attgctgata aatctggagc cggtgagcgt ggttctcgcg gtatcattgc
     4441 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     4501 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     4561 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     4621 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     4681 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     4741 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     4801 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     4861 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     4921 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4981 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5041 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5101 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5161 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5221 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5281 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     5341 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     5401 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     5461 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     5521 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctgat
//
