LOCUS       sequence-133752-        5877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of secreted N-terminally 8His-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 5877)
  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 5877)
  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-3
FEATURES             Location/Qualifiers
     source          1..5877
                     /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..1880
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3012..3029
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3057..3080
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3099..3125
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3234..3253)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3299..3354
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3353..3372)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3713..3729)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3713..3729)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3726..3748)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3737..3753
                     /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(3761..3767,3768..3785,3786..3791))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3806..3827
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3956..3973)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4127..4715)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4207..4226)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4886..5677,5678..5746))
                     /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     5509..5528
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5747..5851)
                     /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 ccatcaccat aaaaccgaag aaggtaaact ggtaatctgg attaacggcg
     1921 ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat accggaatta
     1981 aagtcaccgt tgagcatccc gataaactgg aagagaaatt cccacaggtt gcggcaactg
     2041 gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac gctcaatctg
     2101 gcctgttggc tgaaatcacc ccggccgcag cgttccagga caagctgtat ccgtttacct
     2161 gggatgccgt gcgctacaac ggcaagctga ttgcttaccc gatcgctgtt gaagcgttgt
     2221 ccctgattta taacaaggat ctgctgccga acccgccaaa aacctgggaa gagatcccgg
     2281 cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac
     2341 cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag tatgcagccg
     2401 gaaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct
     2461 tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac tccatcgcag
     2521 aacatgcctt taatcatggc gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca
     2581 acatcgacac cagcgcagtg aattatggtg taacggtgct gccgaccttc aagggtcaac
     2641 catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt ccgaacaaag
     2701 agctggcaaa agagttcctc gaaaactacc tgctgactga tgaaggtctg gaagcggtta
     2761 ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag ttggtgaaag
     2821 atccacgtgt tgccgccaca atggaaaacg cccagaaagg tgaaatcatg cccaacatcc
     2881 cgcagatgtc cgctttctgg tatgccgtgc gcactgcggt gattaacgcc gccagcggtc
     2941 gtcagactgt cgatgcagcc ctggcagccg cgcagactaa tgcggccgcc gcgagctcgg
     3001 ctagcctcga gcaccatcac catcaccatt gataaacgcg tttaattaac cccgggtgga
     3061 gccaccccca gttcgagaag tgataagatg atcaaggata tccctacgac gtgcccgact
     3121 acgcctgata atctagaact agtatcgcga taattcactc ctcaggtgca ggctgcctat
     3181 cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca aataccactg agatcttttt
     3241 ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac ttctggctaa
     3301 taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga
     3361 aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt ttagagtttg
     3421 gcaacatatg ccatatgctg gctgccatga acaaaggtgg ctataaagag gtcatcagta
     3481 tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga cttgaggtta
     3541 gatttttttt atattttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt
     3601 acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc
     3661 tcttctctta tgaagatccc tcgacctgca gcccaagctt ggcgtaatca tggtcatagc
     3721 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     3781 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     3841 cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg gatcgatccg ctgcattaat
     3901 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     3961 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4021 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4081 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4141 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4201 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4261 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4321 aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4381 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4441 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4501 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4561 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4621 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4681 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4741 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     4801 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     4861 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     4921 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     4981 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5041 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5101 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5161 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5221 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5281 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5341 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5401 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5461 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     5521 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     5581 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     5641 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     5701 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     5761 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     5821 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctg
//
