LOCUS       sequence-271261-        4821 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protease Switch Clone ID: 1-A9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4821)
  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 4821)
  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(TP11)-FRB(GPG)-TVMV
FEATURES             Location/Qualifiers
     source          1..4821
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      143..730
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     632..651
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     950..969
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        950..968
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1018..2121
                     /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     2095..2118
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2134..2154
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2161..2184
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2224..2544
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     terminator      complement(3586..3632)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3670..3697
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     3706..3728
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3741..3759)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3833..3937
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3938..4006,4007..4798)
                     /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(4156..4175)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tatactttga attcaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
       61 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
      121 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
      181 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
      241 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
      301 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
      361 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
      421 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
      481 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
      541 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
      601 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
      661 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atctcgtcag gggggcggag
      721 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
      781 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccaa
      841 ttgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
      901 atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attctagagt aatacgactc
      961 actataggga gaaataattt tgtttaactt taaaaaaaaa aagaaggaga ataatctatg
     1021 gggaaaatcg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt
     1081 ctggctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat
     1141 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt
     1201 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc
     1261 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac
     1321 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa
     1381 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg
     1441 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg
     1501 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa
     1561 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt
     1621 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa
     1681 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa
     1741 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt
     1801 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc
     1861 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg
     1921 ggtgccgtag cgctgaagtc ttacgaggaa gagttggtga aagatccgcg tattgccgcc
     1981 actatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc
     2041 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa
     2101 gccctgaaag acgcgcagac taattcgagc tcggagaacc tgtatttcca gtccagtggc
     2161 tggagccacc cgcagttcga aaagtcagga ggtcgcgaat atgtgcgctt tgcgccgggt
     2221 ccgggggtgc aggtggaaac catttctcca ggcgatggcc gtacgttccc gaagcgcggt
     2281 caaacatgcg tagtccatta caccggtatg ctggaagatg gcaaaaagtt cgattcgtcc
     2341 cgtgaccgca acaaaccctt taagttcatg ttgggaaaac aagaagttat ccgtggctgg
     2401 gaggaaggcg tagcgcaaat gtccgttgga caacgtgcaa aattgacaat ctctcccgac
     2461 tacgcgtatg gagcgaccgg ccatccaggt atcattccac ctcacgctac actggtgttc
     2521 gatgttgaac tgctgaaact ggaaggtacc cctacgccaa ctcctacacc cactgggatt
     2581 ctgtggcacg agatgtggca tgagggcctg gaagaggcat ctcgtctgta ttttggtgaa
     2641 cgcaacgtta aaggtatgtt cgaggttttg gagccacttc acgctatgat ggagcgcggt
     2701 ccacagacct tgaaagaaac ctcgtttaat caagcgtatg gccgtgatct gatggaggca
     2761 caggagtggt gtcgtaagta catgaagagt gggaacgtaa aggacctgac tcaggcgtgg
     2821 gatctgtatt accacgtgtt ccgtcgcatt ggtccgggga ccaaggcgct tctgaaaggc
     2881 gtgcgtgact tcaatcctat ttctgcctgt gtatgtttgt tagagaacag ttctgacggc
     2941 cactctgaaa ggctattcgg tataggcttc ggaccgtaca tcatcgccaa ccaacaccta
     3001 tttagaagaa ataacggcga actgaccatc aaaaccatgc acggtgagtt caaagttaag
     3061 aactccactc agcttcagat gaaacctgtt gaaggaagag atattatagt gattaagatg
     3121 gcaaaagact ttccgccatt tccacaaaaa ttgaaatttc gtcagccgac cataaaagat
     3181 agagtttgta tggtaagtac taactttcaa cagaaatcag tctcaagtct tgttagcgaa
     3241 agtagtcaca ttgtgcacaa agaggatact agcttttggc agcactggat aactacgaaa
     3301 gacggtcagt gtggtagtcc attggtatca attatagacg gaaatatatt aggtatccac
     3361 agtctaactc atactaccaa tggttccaat tatttcgtcg agtttcctga aaagtttgtg
     3421 gcgacttatc tggacgcagc cgacggatgg tgcaaaaact ggaagtttaa cgcagacaaa
     3481 ataagttggg gatcattcac cttagttgag gatgcaccgg aggacggcat tgcataaagc
     3541 cccgacagta aaaagagacc ctgactagcc gattccaggc atcaaataaa acgaaaggcc
     3601 cagtctttcg actgagcctt tcgttttatc tgttgtttgt cggtgaacgc tctctactag
     3661 agtcacactg gctcaccttc gggtgggcct ttctgcgttt atactccggg agctgcatgt
     3721 gtcagaggtt taacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     3781 aataatggtt tcttagacgt caggtggcag gcttactttt cggggaaatg tgcgcggaac
     3841 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     3901 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     3961 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     4021 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     4081 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     4141 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     4201 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     4261 aaagcatctt acggatggca tgacagtaag agaattatgt agtgctgcca taaccatgag
     4321 tgataacaca gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     4381 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     4441 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcgatgg caacaacgtt
     4501 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     4561 gatggaggcg gataaagttg caggaccact tcttcgctca gcacttccag ctggttggtt
     4621 tattgctgat aaatctggag ccggtgagcg tggatctcgc ggtatcatag cagcactggg
     4681 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     4741 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     4801 gtcagaccaa gtttactcat a
//
