LOCUS       sequence-417153-        4836 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of recombinant proteins with N-terminal MBP-tag and 
            C-terminal Hisx6-tag in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4836)
  AUTHORS   Poti AL, Denes L, Papp K, Bato C, Banoczi Z, Remenyi A, Alexa A
  TITLE     Phosphorylation-Assisted Luciferase Complementation Assay Designed 
            to Monitor Kinase Activity and Kinase-Domain-Mediated 
            Protein-Protein Binding.
  JOURNAL   Int J Mol Sci. 2023 Oct 3;24(19):14854. doi: 10.3390/ijms241914854.
  PUBMED    37834301
REFERENCE   2  (bases 1 to 4836)
  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: "Int J Mol 
            Sci."; date: "2023-10-3"; volume: "24(19):14854. doi"; pages: " 
            10.3390/ijms241914854"
COMMENT     Sequence Label: pet-MBP
FEATURES             Location/Qualifiers
     source          1..4836
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      15..470
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(102..121)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     312..333
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        496..600
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(601..669,670..1461)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(819..838)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1632..2220
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2121..2140
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2374..2391
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2406..2545
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2471..2490)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2631..2653
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2647..2838)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3347..3366
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3347..3365
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3427..4527
                     /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
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      4384..4393
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     4501..4524
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             4585..4605
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4624..4641
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             4648..4665
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4683..4700
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4753..4771)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4767..4814
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
       61 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
      121 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
      181 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
      241 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
      301 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
      361 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
      421 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
      481 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
      541 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
      601 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
      661 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
      721 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
      781 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
      841 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
      901 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
      961 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     1021 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     1081 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     1141 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     1201 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     1261 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     1321 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     1381 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     1441 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     1501 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     1561 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     1621 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     1681 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     1741 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     1801 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     1861 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     1921 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     1981 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     2041 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     2101 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     2161 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     2221 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     2281 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     2341 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     2401 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     2461 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     2521 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     2581 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     2641 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     2701 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     2761 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     2821 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     2881 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     2941 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     3001 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     3061 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     3121 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     3181 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     3241 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     3301 ccgtggccag gacccaacgc tgcccgagat ctcgatcccg cgaaattaat acgactcact
     3361 atagggagac cacaacggtt tccctctaga ttttcacgag cacttcacca acaaggacca
     3421 tagattatga aaactgaaga aggtaaactg gtaatctgga ttaacggcga taaaggctat
     3481 aacggtctcg ctgaagtcgg taagaaattc gagaaagata ccggaattaa agtcaccgtt
     3541 gagcatccgg ataaactgga agagaaattc ccacaggttg cggcaactgg cgatggccct
     3601 gacattatct tctgggcaca cgaccgcttt ggtggctacg ctcaatctgg cctgttggct
     3661 gaaatcaccc cggacaaagc gttccaggac aagctgtatc cgtttacctg ggatgccgta
     3721 cgttacaacg gcaagctgat tgcttacccg atcgctgttg aagcgttatc gctgatttat
     3781 aacaaagatc tgctgccgaa cccgccaaaa acctgggaag agatcccggc gctggataaa
     3841 gaactgaaag cgaaaggtaa gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc
     3901 tggccgctga ttgctgctga cgggggttat gcgttcaagt atgaaaacgg caagtacgac
     3961 attaaagacg tgggcgtgga taacgctggc gcgaaagcgg gtctgacctt cctggttgac
     4021 ctgattaaaa acaaacacat gaatgcagac accgattact ccatcgcaga agctgccttt
     4081 aataaaggcg aaacagcgat gaccatcaac ggcccgtggg catggtccaa catcgacacc
     4141 agcaaagtga attatggtgt aacggtactg ccgaccttca agggtcaacc atccaaaccg
     4201 ttcgttggcg tgctgagcgc aggtattaac gccgccagtc cgaacaaaga gctggcaaaa
     4261 gagttcctcg aaaactatct gctgactgat gaaggtctgg aagcggttaa taaagacaaa
     4321 ccgctgggtg ccgtagcgct gaagtcttac gaggaagagt tggcgaaaga tccacgtatt
     4381 gccgccacca tggaaaacgc ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc
     4441 gctttctggt atgccgtgcg tactgcggtg atcaacgccg ccagcggtcg tcagactgtc
     4501 gatgaagccc tgaaagacgc gcagactaat tcgagctcga acaacaacac tagtggctcc
     4561 ggtggtggtg gcggttccat gagcgagaat ctttattttc agggatccgc tagcgctgtc
     4621 gacctggttc cgcgtggcag cggccgccac catcaccacc accactgagg gccgcactcg
     4681 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
     4741 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     4801 tgaggggttt tttgctgaaa ggaggaacta tatccg
//
