LOCUS       sequence-133754-        5751 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of cytoplasmic 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 5751)
  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 5751)
  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-6
FEATURES             Location/Qualifiers
     source          1..5751
                     /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             2886..2903
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2931..2954
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2973..2999
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3108..3127)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3173..3228
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3227..3246)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3587..3603)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3587..3603)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3600..3622)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3611..3627
                     /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(3635..3641,3642..3659,3660..3665))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3680..3701
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3830..3847)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4001..4589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4081..4100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4760..5551,5552..5620))
                     /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     5383..5402
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5621..5725)
                     /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 ctaatgcggc cgccgcgagc tcggctagcc
     2881 tcgagcacca tcaccatcac cattgataaa cgcgtttaat taaccccggg tggagccacc
     2941 cccagttcga gaagtgataa gatgatcaag gatatcccta cgacgtgccc gactacgcct
     3001 gataatctag aactagtatc gcgataattc actcctcagg tgcaggctgc ctatcagaag
     3061 gtggtggctg gtgtggccaa tgccctggct cacaaatacc actgagatct ttttccctct
     3121 gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg
     3181 aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca
     3241 tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca
     3301 tatgccatat gctggctgcc atgaacaaag gtggctataa agaggtcatc agtatatgaa
     3361 acagccccct gctgtccatt ccttattcca tagaaaagcc ttgacttgag gttagatttt
     3421 ttttatattt tgttttgtgt tatttttttc tttaacatcc ctaaaatttt ccttacatgt
     3481 tttactagcc agatttttcc tcctctcctg actactccca gtcatagctg tccctcttct
     3541 cttatgaaga tccctcgacc tgcagcccaa gcttggcgta atcatggtca tagctgtttc
     3601 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3661 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     3721 ccgctttcca gtcgggaaac ctgtcgtgcc agcggatcga tccgctgcat taatgaatcg
     3781 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     3841 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3901 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3961 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4021 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4081 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4141 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct
     4201 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4261 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4321 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4381 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4441 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4501 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4561 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4621 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4681 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4741 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4801 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4861 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4921 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4981 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5041 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5101 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5161 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5221 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5281 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5341 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5401 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5461 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5521 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5581 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5641 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5701 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct g
//
