LOCUS       sequence-132066-        5856 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of secreted mMBP-fused 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 5856)
  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 5856)
  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-1
FEATURES             Location/Qualifiers
     source          1..5856
                     /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             2991..3008
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3036..3059
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3078..3104
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3213..3232)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3278..3333
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3332..3351)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3692..3708)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3692..3708)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3705..3727)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3716..3732
                     /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(3740..3746,3747..3764,3765..3770))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3785..3806
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3935..3952)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4106..4694)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4186..4205)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4865..5656,5657..5725))
                     /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     5488..5507
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5726..5830)
                     /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 gcggccgccg cgagctcggc tagcctcgag caccatcacc
     3001 atcaccattg ataaacgcgt ttaattaacc ccgggtggag ccacccccag ttcgagaagt
     3061 gataagatga tcaaggatat ccctacgacg tgcccgacta cgcctgataa tctagaacta
     3121 gtatcgcgat aattcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg
     3181 gccaatgccc tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg
     3241 gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt
     3301 gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat
     3361 catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc catatgctgg
     3421 ctgccatgaa caaaggtggc tataaagagg tcatcagtat atgaaacagc cccctgctgt
     3481 ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt
     3541 tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac tagccagatt
     3601 tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat gaagatccct
     3661 cgacctgcag cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     3721 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     3781 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3841 gaaacctgtc gtgccagcgg atcgatccgc tgcattaatg aatcggccaa cgcgcgggga
     3901 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3961 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4021 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4081 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4141 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4201 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4261 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc
     4321 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4381 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4441 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4501 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     4561 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4621 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4681 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4741 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4801 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     4861 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     4921 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     4981 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5041 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5101 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5161 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5221 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5281 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5341 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5401 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5461 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     5521 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5581 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     5641 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     5701 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     5761 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     5821 gggttccgcg cacatttccc cgaaaagtgc cacctg
//
