LOCUS       sequence-133760-        5910 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of secreted mMBP TEV cleavage site-linked 
            fusion proteins with C-terminal 6His-tag/Strep-tag II/HA-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5910)
  AUTHORS   Bokhove M, Sadat Al Hosseini H, Saito T, Dioguardi E, 
            Gegenschatz-Schmid K, Nishimura K, Raj I, de Sanctis D, Han L, 
            Jovine L
  TITLE     Easy mammalian expression and crystallography of maltose-binding 
            protein-fused human proteins.
  JOURNAL   J Struct Biol. 2016 Feb 3. 194:1-7. pii: S1047-8477(16)30015-6. doi:
            10.1016/j.jsb.2016.01.016.
  PUBMED    26850170
REFERENCE   2  (bases 1 to 5910)
  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: "J Struct 
            Biol. 2016 Feb 3. 194:1-7. pii: S1047-8477(16)30015-6. doi: 
            10.1016/j.jsb.2016.01.016."
COMMENT     Sequence Label: pHLmMBP-11
FEATURES             Location/Qualifiers
     source          1..5910
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1594..1616
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     regulatory      1758..1767
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1860..2957
                     /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
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPAAAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYAAGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEHAFNHGET
                     AMTINGPWAWSNIDTSAVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRVAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDAALAAAQT"
     CDS             2994..3014
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3045..3062
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3090..3113
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3132..3158
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3267..3286)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3332..3387
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3386..3405)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3746..3762)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3746..3762)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3759..3781)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3770..3786
                     /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)."
     promoter        complement(join(3794..3800,3801..3818,3819..3824))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3839..3860
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3989..4006)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4160..4748)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4240..4259)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4919..5710,5711..5779))
                     /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     5542..5561
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5780..5884)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attgcggccg tctcaggccg
     1741 aattcgtcga caagcttgcc accatgggga tccttcccag ccctgggatg cctgcgctgc
     1801 tctccctcgt gagccttctc tccgtgctgc tgatgggttg cgtagctgaa accggtacca
     1861 aaatcgaaga aggtaaactg gtaatctgga ttaacggcga taaaggctat aacggtctcg
     1921 ctgaagtcgg taagaaattc gagaaagata ccggaattaa agtcaccgtt gagcatcccg
     1981 ataaactgga agagaaattc ccacaggttg cggcaactgg cgatggccct gacattatct
     2041 tctgggcaca cgaccgcttt ggtggctacg ctcaatctgg cctgttggct gaaatcaccc
     2101 cggccgcagc gttccaggac aagctgtatc cgtttacctg ggatgccgtg cgctacaacg
     2161 gcaagctgat tgcttacccg atcgctgttg aagcgttgtc cctgatttat aacaaggatc
     2221 tgctgccgaa cccgccaaaa acctgggaag agatcccggc gctggataaa gaactgaaag
     2281 cgaaaggtaa gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc tggccgctga
     2341 ttgctgctga cgggggttat gcgttcaagt atgcagccgg aaagtacgac attaaagacg
     2401 tgggcgtgga taacgctggc gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa
     2461 acaaacacat gaatgcagac accgattact ccatcgcaga acatgccttt aatcatggcg
     2521 aaacagcgat gaccatcaac ggcccgtggg catggtccaa catcgacacc agcgcagtga
     2581 attatggtgt aacggtgctg ccgaccttca agggtcaacc atccaaaccg ttcgttggcg
     2641 tgctgagcgc aggtattaac gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg
     2701 aaaactacct gctgactgat gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg
     2761 ccgtagcgct gaagtcttac gaggaagagt tggtgaaaga tccacgtgtt gccgccacaa
     2821 tggaaaacgc ccagaaaggt gaaatcatgc ccaacatccc gcagatgtcc gctttctggt
     2881 atgccgtgcg cactgcggtg attaacgccg ccagcggtcg tcagactgtc gatgcagccc
     2941 tggcagccgc gcagactaat atcaccaagg gcggagggag cggcggaggg agcgaaaacc
     3001 tgtattttca gagcgcggcc gccgcgagct cggctagcct cgagcaccat caccatcacc
     3061 attgataaac gcgtttaatt aaccccgggt ggagccaccc ccagttcgag aagtgataag
     3121 atgatcaagg atatccctac gacgtgcccg actacgcctg ataatctaga actagtatcg
     3181 cgataattca ctcctcaggt gcaggctgcc tatcagaagg tggtggctgg tgtggccaat
     3241 gccctggctc acaaatacca ctgagatctt tttccctctg ccaaaaatta tggggacatc
     3301 atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt cattgcaata
     3361 gtgtgttgga attttttgtg tctctcactc ggaaggacat atgggagggc aaatcattta
     3421 aaacatcaga atgagtattt ggtttagagt ttggcaacat atgccatatg ctggctgcca
     3481 tgaacaaagg tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc
     3541 cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt
     3601 atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct
     3661 cctctcctga ctactcccag tcatagctgt ccctcttctc ttatgaagat ccctcgacct
     3721 gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3781 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     3841 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3901 tgtcgtgcca gcggatcgat ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3961 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4021 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4081 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4141 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4201 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4261 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4321 cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt
     4381 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4441 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4501 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4561 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     4621 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4681 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4741 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4801 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4861 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4921 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4981 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5041 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5101 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5161 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5221 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5281 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5341 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5401 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5461 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5521 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5581 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5641 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5701 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5761 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5821 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5881 cgcgcacatt tccccgaaaa gtgccacctg
//
