LOCUS       sequence-271259-        4815 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protease Switch Clone ID: 2-B3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4815)
  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 4815)
  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(P9)-FRB(GPG)-TVMV
FEATURES             Location/Qualifiers
     source          1..4815
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(128..147)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      936..1523
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1425..1444
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1743..1762
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1743..1761
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1811..2914
                     /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     2888..2911
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2927..2947
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2954..2977
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3017..3337
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     terminator      complement(4373..4419)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4457..4484
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     4493..4515
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4528..4546)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4620..4724
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4725..4793,4794..4815,1..770)
                     /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"
ORIGIN
        1 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
       61 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
      121 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
      181 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
      241 ttacggatgg catgacagta agagaattat gtagtgctgc cataaccatg agtgataaca
      301 cagcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
      361 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
      421 taccaaacga cgagcgtgac accacgatgc ctgtagcgat ggcaacaacg ttgcgcaaac
      481 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
      541 cggataaagt tgcaggacca cttcttcgct cagcacttcc agctggttgg tttattgctg
      601 ataaatctgg agccggtgag cgtggatctc gcggtatcat agcagcactg gggccagatg
      661 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
      721 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
      781 aagtttactc atatatactt tgaattcaaa cttcattttt aatttaaaag gatctaggtg
      841 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
      901 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
      961 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     1021 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     1081 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     1141 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     1201 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     1261 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     1321 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     1381 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     1441 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatctcgt
     1501 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     1561 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     1621 gtattaccgc caattgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     1681 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattctag
     1741 agtaatacga ctcactatag ggagaaataa ttttgtttaa ctttaaaaaa aaaaagaagg
     1801 agaataatct atggggaaaa tcgaagaagg taaactggta atctggatta acggcgataa
     1861 aggctataac ggtctggctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt
     1921 caccgttgag catccggata aactggaaga gaaattccca caggttgcgg caactggcga
     1981 tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct
     2041 gttggctgaa atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga
     2101 tgccgtacgt tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct
     2161 gatttataac aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct
     2221 ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta
     2281 cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa
     2341 gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct
     2401 ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc
     2461 tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat
     2521 cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc
     2581 caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct
     2641 ggcaaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa
     2701 agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg tgaaagatcc
     2761 gcgtattgcc gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca
     2821 gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca
     2881 gactgtcgat gaagccctga aagacgcgca gactaattcg agctcggaga acctgtattt
     2941 ccagtccagt ggctggagcc acccgcagtt cgaaaagtca ggaggtcgcg aatatgtgcg
     3001 ctttgcgccg ggtccggggg tgcaggtgga aaccatttct ccaggcgatg gccgtacgtt
     3061 cccgaagcgc ggtcaaacat gcgtagtcca ttacaccggt atgctggaag atggcaaaaa
     3121 gttcgattcg tcccgtgacc gcaacaaacc ctttaagttc atgttgggaa aacaagaagt
     3181 tatccgtggc tgggaggaag gcgtagcgca aatgtccgtt ggacaacgtg caaaattgac
     3241 aatctctccc gactacgcgt atggagcgac cggccatcca ggtatcattc cacctcacgc
     3301 tacactggtg ttcgatgttg aactgctgaa actggaaggt ccgcctccac cacctccgcc
     3361 agggattctg tggcacgaga tgtggcatga gggcctggaa gaggcatctc gtctgtattt
     3421 tggtgaacgc aacgttaaag gtatgttcga ggttttggag ccacttcacg ctatgatgga
     3481 gcgcggtcca cagaccttga aagaaacctc gtttaatcaa gcgtatggcc gtgatctgat
     3541 ggaggcacag gagtggtgtc gtaagtacat gaagagtggg aacgtaaagg acctgactca
     3601 ggcgtgggat ctgtattacc acgtgttccg tcgcattggt ccggggtcca aggcgcttct
     3661 gaaaggcgtg cgtgacttca atcctatttc tgcctgtgta tgtttgttag agaacagttc
     3721 tgacggccac tctgaaaggc tattcggtat aggcttcgga ccgtacatca tcgccaacca
     3781 acacctattt agaagaaata acggcgaact gaccatcaaa accatgcacg gtgagttcaa
     3841 agttaagaac tccactcagc ttcagatgaa acctgttgaa ggaagagata ttatagtgat
     3901 taagatggca aaagactttc cgccatttcc acaaaaattg aaatttcgtc agccgaccat
     3961 aaaagataga gtttgtatgg taagtactaa ctttcaacag aaatcagtct caagtcttgt
     4021 tagcgaaagt agtcacattg tgcacaaaga ggatactagc ttttggcagc actggataac
     4081 tacgaaagac ggtcagtgtg gtagtccatt ggtatcaatt atagacggaa atatattagg
     4141 tatccacagt ctaactcata ctaccaatgg ttccaattat ttcgtcgagt ttcctgaaaa
     4201 gtttgtggcg acttatctgg acgcagccga cggatggtgc aaaaactgga agtttaacgc
     4261 agacaaaata agttggggat cattcacctt agttgaggat gcaccggagg acggcattgc
     4321 ataaagcccc gacagtaaaa agagaccctg actagccgat tccaggcatc aaataaaacg
     4381 aaaggcccag tctttcgact gagcctttcg ttttatctgt tgtttgtcgg tgaacgctct
     4441 ctactagagt cacactggct caccttcggg tgggcctttc tgcgtttata ctccgggagc
     4501 tgcatgtgtc agaggtttaa cgaaagggcc tcgtgatacg cctattttta taggttaatg
     4561 tcatgataat aatggtttct tagacgtcag gtggcaggct tacttttcgg ggaaatgtgc
     4621 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4681 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     4741 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     4801 aaacgctggt gaaag
//
