LOCUS       sequence-271258-        4842 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protease Switch Clone ID: 1-C9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4842)
  AUTHORS   Grawe A, Ranglack J, Weyrich A, Stein V
  TITLE     iFLinkC: an iterative functional linker cloning strategy for the 
            combinatorial assembly and recombination of linker peptides with 
            functional domains.
  JOURNAL   Nucleic Acids Res. 2020 Jan 11. pii: 5700546. doi: 
            10.1093/nar/gkz1210.
  PUBMED    31925441
REFERENCE   2  (bases 1 to 4842)
  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: "Nucleic 
            Acids Res. 2020 Jan 11. pii: 5700546. doi: 10.1093/nar/gkz1210."
COMMENT     Sequence Label: pFLinkC-AI(GPG)-FKBP12(EAAAK16)-FRB(PAS5)-TVMV
FEATURES             Location/Qualifiers
     source          1..4842
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     252..271
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     570..589
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        570..588
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             638..1741
                     /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="MGKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKL
                     EEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGK
                     LIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLI
                     AADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKG
                     ETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEF
                     LENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSA
                     FWYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1715..1738
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1754..1774
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1781..1804
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1844..2164
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     terminator      complement(3227..3273)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3311..3338
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     3347..3369
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3382..3400)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3474..3578
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3579..3647,3648..4439)
                     /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     complement(3797..3816)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4605..4842,1..350)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
       61 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
      121 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
      181 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
      241 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
      301 cttgagcgtc gatttttgtg atctcgtcag gggggcggag cctatggaaa aacgccagca
      361 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
      421 cgttatcccc tgattctgtg gataaccgta ttaccgccaa ttgatgtcgg cgatataggc
      481 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
      541 cgagatctcg atcccgcgaa attctagagt aatacgactc actataggga gaaataattt
      601 tgtttaactt taaaaaaaaa aagaaggaga ataatctatg gggaaaatcg aagaaggtaa
      661 actggtaatc tggattaacg gcgataaagg ctataacggt ctggctgaag tcggtaagaa
      721 attcgagaaa gataccggaa ttaaagtcac cgttgagcat ccggataaac tggaagagaa
      781 attcccacag gttgcggcaa ctggcgatgg ccctgacatt atcttctggg cacacgaccg
      841 ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc accccggaca aagcgttcca
      901 ggacaagctg tatccgttta cctgggatgc cgtacgttac aacggcaagc tgattgctta
      961 cccgatcgct gttgaagcgt tatcgctgat ttataacaaa gatctgctgc cgaacccgcc
     1021 aaaaacctgg gaagagatcc cggcgctgga taaagaactg aaagcgaaag gtaagagcgc
     1081 gctgatgttc aacctgcaag aaccgtactt cacctggccg ctgattgctg ctgacggggg
     1141 ttatgcgttc aagtatgaaa acggcaagta cgacattaaa gacgtgggcg tggataacgc
     1201 tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt aaaaacaaac acatgaatgc
     1261 agacaccgat tactccatcg cagaagctgc ctttaataaa ggcgaaacag cgatgaccat
     1321 caacggcccg tgggcatggt ccaacatcga caccagcaaa gtgaattatg gtgtaacggt
     1381 actgccgacc ttcaagggtc aaccatccaa accgttcgtt ggcgtgctga gcgcaggtat
     1441 taacgccgcc agtccgaaca aagagctggc aaaagagttc ctcgaaaact atctgctgac
     1501 tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg ggtgccgtag cgctgaagtc
     1561 ttacgaggaa gagttggtga aagatccgcg tattgccgcc actatggaaa acgcccagaa
     1621 aggtgaaatc atgccgaaca tcccgcagat gtccgctttc tggtatgccg tgcgtactgc
     1681 ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa gccctgaaag acgcgcagac
     1741 taattcgagc tcggagaacc tgtatttcca gtccagtggc tggagccacc cgcagttcga
     1801 aaagtcagga ggtcgcgaat atgtgcgctt tgcgccgggt ccgggggtgc aggtggaaac
     1861 catttctcca ggcgatggcc gtacgttccc gaagcgcggt caaacatgcg tagtccatta
     1921 caccggtatg ctggaagatg gcaaaaagtt cgattcgtcc cgtgaccgca acaaaccctt
     1981 taagttcatg ttgggaaaac aagaagttat ccgtggctgg gaggaaggcg tagcgcaaat
     2041 gtccgttgga caacgtgcaa aattgacaat ctctcccgac tacgcgtatg gagcgaccgg
     2101 ccatccaggt atcattccac ctcacgctac actggtgttc gatgttgaac tgctgaaact
     2161 ggaaggtgga gcagaagcgg ccgctaaaga ggcagctgca aaagcaggag ggattctgtg
     2221 gcacgagatg tggcatgagg gcctggaaga ggcatctcgt ctgtattttg gtgaacgcaa
     2281 cgttaaaggt atgttcgagg ttttggagcc acttcacgct atgatggagc gcggtccaca
     2341 gaccttgaaa gaaacctcgt ttaatcaagc gtatggccgt gatctgatgg aggcacagga
     2401 gtggtgtcgt aagtacatga agagtgggaa cgtaaaggac ctgactcagg cgtgggatct
     2461 gtattaccac gtgttccgtc gcattggttc acctgcaggg tccaaggcgc ttctgaaagg
     2521 cgtgcgtgac ttcaatccta tttctgcctg tgtatgtttg ttagagaaca gttctgacgg
     2581 ccactctgaa aggctattcg gtataggctt cggaccgtac atcatcgcca accaacacct
     2641 atttagaaga aataacggcg aactgaccat caaaaccatg cacggtgagt tcaaagttaa
     2701 gaactccact cagcttcaga tgaaacctgt tgaaggaaga gatattatag tgattaagat
     2761 ggcaaaagac tttccgccat ttccacaaaa attgaaattt cgtcagccga ccataaaaga
     2821 tagagtttgt atggtaagta ctaactttca acagaaatca gtctcaagtc ttgttagcga
     2881 aagtagtcac attgtgcaca aagaggatac tagcttttgg cagcactgga taactacgaa
     2941 agacggtcag tgtggtagtc cattggtatc aattatagac ggaaatatat taggtatcca
     3001 cagtctaact catactacca atggttccaa ttatttcgtc gagtttcctg aaaagtttgt
     3061 ggcgacttat ctggacgcag ccgacggatg gtgcaaaaac tggaagttta acgcagacaa
     3121 aataagttgg ggatcattca ccttagttga ggatgcaccg gaggacggca ttgcataaag
     3181 ccccgacagt aaaaagagac cctgactagc cgattccagg catcaaataa aacgaaaggc
     3241 ccagtctttc gactgagcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta
     3301 gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tatactccgg gagctgcatg
     3361 tgtcagaggt ttaacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     3421 taataatggt ttcttagacg tcaggtggca ggcttacttt tcggggaaat gtgcgcggaa
     3481 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3541 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     3601 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     3661 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     3721 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     3781 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     3841 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     3901 aaaagcatct tacggatggc atgacagtaa gagaattatg tagtgctgcc ataaccatga
     3961 gtgataacac agcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     4021 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     4081 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcgatg gcaacaacgt
     4141 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     4201 ggatggaggc ggataaagtt gcaggaccac ttcttcgctc agcacttcca gctggttggt
     4261 ttattgctga taaatctgga gccggtgagc gtggatctcg cggtatcata gcagcactgg
     4321 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     4381 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     4441 tgtcagacca agtttactca tatatacttt gaattcaaac ttcattttta atttaaaagg
     4501 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     4561 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4621 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4681 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4741 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4801 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ct
//
