LOCUS       sequence-133755-        5796 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of cytoplasmic 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 5796)
  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 5796)
  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-8
FEATURES             Location/Qualifiers
     source          1..5796
                     /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"
     CDS             1752..2852
                     /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="MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPAAAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYAAGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEHAFNHGE
                     TAMTINGPWAWSNIDTSAVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRVAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDAALAAAQT"
     CDS             2880..2900
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2931..2948
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2976..2999
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3018..3044
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3153..3172)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3218..3273
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3272..3291)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3632..3648)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3632..3648)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3645..3667)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3656..3672
                     /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(3680..3686,3687..3704,3705..3710))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3725..3746
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3875..3892)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4046..4634)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4126..4145)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4805..5596,5597..5665))
                     /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     5428..5447
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5666..5770)
                     /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 aattcgccac catgaaaact gaagaaggta aactggtaat ctggattaac ggcgataaag
     1801 gctataacgg tctcgctgaa gtcggtaaga aattcgagaa agataccgga attaaagtca
     1861 ccgttgagca tcccgataaa ctggaagaga aattcccaca ggttgcggca actggcgatg
     1921 gccctgacat tatcttctgg gcacacgacc gctttggtgg ctacgctcaa tctggcctgt
     1981 tggctgaaat caccccggcc gcagcgttcc aggacaagct gtatccgttt acctgggatg
     2041 ccgtgcgcta caacggcaag ctgattgctt acccgatcgc tgttgaagcg ttgtccctga
     2101 tttataacaa ggatctgctg ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg
     2161 ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact
     2221 tcacctggcc gctgattgct gctgacgggg gttatgcgtt caagtatgca gccggaaagt
     2281 acgacattaa agacgtgggc gtggataacg ctggcgcgaa agcgggtctg accttcctgg
     2341 ttgacctgat taaaaacaaa cacatgaatg cagacaccga ttactccatc gcagaacatg
     2401 cctttaatca tggcgaaaca gcgatgacca tcaacggccc gtgggcatgg tccaacatcg
     2461 acaccagcgc agtgaattat ggtgtaacgg tgctgccgac cttcaagggt caaccatcca
     2521 aaccgttcgt tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac aaagagctgg
     2581 caaaagagtt cctcgaaaac tacctgctga ctgatgaagg tctggaagcg gttaataaag
     2641 acaaaccgct gggtgccgta gcgctgaagt cttacgagga agagttggtg aaagatccac
     2701 gtgttgccgc cacaatggaa aacgcccaga aaggtgaaat catgcccaac atcccgcaga
     2761 tgtccgcttt ctggtatgcc gtgcgcactg cggtgattaa cgccgccagc ggtcgtcaga
     2821 ctgtcgatgc agccctggca gccgcgcaga ctaatggcgg agggagcggc ggagggagcg
     2881 agaatcttta ttttcagggt gcggccgccg cgagctcggc tagcctcgag caccatcacc
     2941 atcaccattg ataaacgcgt ttaattaacc ccgggtggag ccacccccag ttcgagaagt
     3001 gataagatga tcaaggatat ccctacgacg tgcccgacta cgcctgataa tctagaacta
     3061 gtatcgcgat aattcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg
     3121 gccaatgccc tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg
     3181 gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt
     3241 gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat
     3301 catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc catatgctgg
     3361 ctgccatgaa caaaggtggc tataaagagg tcatcagtat atgaaacagc cccctgctgt
     3421 ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt
     3481 tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac tagccagatt
     3541 tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat gaagatccct
     3601 cgacctgcag cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     3661 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     3721 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3781 gaaacctgtc gtgccagcgg atcgatccgc tgcattaatg aatcggccaa cgcgcgggga
     3841 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3901 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3961 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4021 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4081 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4141 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4201 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc
     4261 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4321 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4381 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4441 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     4501 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4561 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4621 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4681 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4741 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     4801 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     4861 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     4921 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     4981 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5041 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5101 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5161 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5221 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5281 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5341 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5401 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     5461 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5521 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     5581 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     5641 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     5701 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     5761 gggttccgcg cacatttccc cgaaaagtgc cacctg
//
