LOCUS       sequence-132067-        5871 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of secreted N-terminally 6His-tagged 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 5871)
  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 5871)
  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-2
FEATURES             Location/Qualifiers
     source          1..5871
                     /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             1857..1874
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3006..3023
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3051..3074
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3093..3119
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3228..3247)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3293..3348
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3347..3366)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3707..3723)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3707..3723)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3720..3742)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3731..3747
                     /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(3755..3761,3762..3779,3780..3785))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3800..3821
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3950..3967)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4121..4709)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4201..4220)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4880..5671,5672..5740))
                     /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     5503..5522
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5741..5845)
                     /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 accggtcacc
     1861 atcaccatca ccataaaacc gaagaaggta aactggtaat ctggattaac ggcgataaag
     1921 gctataacgg tctcgctgaa gtcggtaaga aattcgagaa agataccgga attaaagtca
     1981 ccgttgagca tcccgataaa ctggaagaga aattcccaca ggttgcggca actggcgatg
     2041 gccctgacat tatcttctgg gcacacgacc gctttggtgg ctacgctcaa tctggcctgt
     2101 tggctgaaat caccccggcc gcagcgttcc aggacaagct gtatccgttt acctgggatg
     2161 ccgtgcgcta caacggcaag ctgattgctt acccgatcgc tgttgaagcg ttgtccctga
     2221 tttataacaa ggatctgctg ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg
     2281 ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact
     2341 tcacctggcc gctgattgct gctgacgggg gttatgcgtt caagtatgca gccggaaagt
     2401 acgacattaa agacgtgggc gtggataacg ctggcgcgaa agcgggtctg accttcctgg
     2461 ttgacctgat taaaaacaaa cacatgaatg cagacaccga ttactccatc gcagaacatg
     2521 cctttaatca tggcgaaaca gcgatgacca tcaacggccc gtgggcatgg tccaacatcg
     2581 acaccagcgc agtgaattat ggtgtaacgg tgctgccgac cttcaagggt caaccatcca
     2641 aaccgttcgt tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac aaagagctgg
     2701 caaaagagtt cctcgaaaac tacctgctga ctgatgaagg tctggaagcg gttaataaag
     2761 acaaaccgct gggtgccgta gcgctgaagt cttacgagga agagttggtg aaagatccac
     2821 gtgttgccgc cacaatggaa aacgcccaga aaggtgaaat catgcccaac atcccgcaga
     2881 tgtccgcttt ctggtatgcc gtgcgcactg cggtgattaa cgccgccagc ggtcgtcaga
     2941 ctgtcgatgc agccctggca gccgcgcaga ctaatgcggc cgccgcgagc tcggctagcc
     3001 tcgagcacca tcaccatcac cattgataaa cgcgtttaat taaccccggg tggagccacc
     3061 cccagttcga gaagtgataa gatgatcaag gatatcccta cgacgtgccc gactacgcct
     3121 gataatctag aactagtatc gcgataattc actcctcagg tgcaggctgc ctatcagaag
     3181 gtggtggctg gtgtggccaa tgccctggct cacaaatacc actgagatct ttttccctct
     3241 gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg
     3301 aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca
     3361 tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca
     3421 tatgccatat gctggctgcc atgaacaaag gtggctataa agaggtcatc agtatatgaa
     3481 acagccccct gctgtccatt ccttattcca tagaaaagcc ttgacttgag gttagatttt
     3541 ttttatattt tgttttgtgt tatttttttc tttaacatcc ctaaaatttt ccttacatgt
     3601 tttactagcc agatttttcc tcctctcctg actactccca gtcatagctg tccctcttct
     3661 cttatgaaga tccctcgacc tgcagcccaa gcttggcgta atcatggtca tagctgtttc
     3721 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3781 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     3841 ccgctttcca gtcgggaaac ctgtcgtgcc agcggatcga tccgctgcat taatgaatcg
     3901 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     3961 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4021 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4081 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4141 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4201 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4261 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct
     4321 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4381 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4441 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4501 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4561 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4621 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4681 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4741 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4801 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4861 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4921 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4981 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5041 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5101 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5161 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5221 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5281 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5341 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5401 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5461 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5521 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5581 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5641 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5701 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5761 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5821 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct g
//
