LOCUS       sequence-271262-        4776 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protease Switch Clone ID: 2-A5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4776)
  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 4776)
  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(G3)-PDZ(GPG-G)FN3(GGS4)-TVMV
FEATURES             Location/Qualifiers
     source          1..4776
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     208..227
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        208..226
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             276..1379
                     /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     1353..1376
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1392..1412
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1419..1442
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     terminator      complement(2799..2845)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2883..2910
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     2919..2941
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2954..2972)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3046..3150
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3151..3219,3220..4011)
                     /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(3369..3388)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4177..4764
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4666..4685
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
       61 ttatcccctg attctgtgga taaccgtatt accgccaatt gatgtcggcg atataggcgc
      121 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
      181 agatctcgat cccgcgaaat tctagagtaa tacgactcac tatagggaga aataattttg
      241 tttaacttta aaaaaaaaaa gaaggagaat aatctatggg gaaaatcgaa gaaggtaaac
      301 tggtaatctg gattaacggc gataaaggct ataacggtct ggctgaagtc ggtaagaaat
      361 tcgagaaaga taccggaatt aaagtcaccg ttgagcatcc ggataaactg gaagagaaat
      421 tcccacaggt tgcggcaact ggcgatggcc ctgacattat cttctgggca cacgaccgct
      481 ttggtggcta cgctcaatct ggcctgttgg ctgaaatcac cccggacaaa gcgttccagg
      541 acaagctgta tccgtttacc tgggatgccg tacgttacaa cggcaagctg attgcttacc
      601 cgatcgctgt tgaagcgtta tcgctgattt ataacaaaga tctgctgccg aacccgccaa
      661 aaacctggga agagatcccg gcgctggata aagaactgaa agcgaaaggt aagagcgcgc
      721 tgatgttcaa cctgcaagaa ccgtacttca cctggccgct gattgctgct gacgggggtt
      781 atgcgttcaa gtatgaaaac ggcaagtacg acattaaaga cgtgggcgtg gataacgctg
      841 gcgcgaaagc gggtctgacc ttcctggttg acctgattaa aaacaaacac atgaatgcag
      901 acaccgatta ctccatcgca gaagctgcct ttaataaagg cgaaacagcg atgaccatca
      961 acggcccgtg ggcatggtcc aacatcgaca ccagcaaagt gaattatggt gtaacggtac
     1021 tgccgacctt caagggtcaa ccatccaaac cgttcgttgg cgtgctgagc gcaggtatta
     1081 acgccgccag tccgaacaaa gagctggcaa aagagttcct cgaaaactat ctgctgactg
     1141 atgaaggtct ggaagcggtt aataaagaca aaccgctggg tgccgtagcg ctgaagtctt
     1201 acgaggaaga gttggtgaaa gatccgcgta ttgccgccac tatggaaaac gcccagaaag
     1261 gtgaaatcat gccgaacatc ccgcagatgt ccgctttctg gtatgccgtg cgtactgcgg
     1321 tgatcaacgc cgccagcggt cgtcagactg tcgatgaagc cctgaaagac gcgcagacta
     1381 attcgagctc ggagaacctg tatttccagt ccagtggctg gagccacccg cagttcgaaa
     1441 agtcaggagg tcgcgaatat gtgcgctttg cgccgggtgg agggtcaccc gagttggggt
     1501 tcagtatttc tggcggtgtt gggggtcgtg gtaatccctt ccgccctgac gatgacggaa
     1561 tctttgtcac ccgtgtgcaa cccgaagggc ctgcaagtaa gttgttgcag ccaggcgata
     1621 agatcattca ggctaatggc tattctttca tcaatattga gcatggtcaa gcggtatcct
     1681 tattgaaaac ttttcaaaac acagtggaac ttatcattgt gcgtgaggtg ggtaacggcg
     1741 ctaagcaaga gatccgtgtc cgtgtcgaaa aggatggtcc ggggggggtt agttcggtgc
     1801 ctacaaattt ggaggtggtt gcggcaacac ccacctcctt gctgatctct tgggacgcct
     1861 accgtgagtt acccgtgtcc tactaccgta tcacgtacgg tgagactggc gggaactcac
     1921 ctgtgcagga atttacagtg ccggggtcca aatcaaccgc taccatctcg ggattgaagc
     1981 cgggtgttga ttatactatc actgtgtatg cccactataa ctaccactac tatagttccc
     2041 ccatctccat caactatcgc ggtggaagcg ggtccaaggc gcttctgaaa ggcgtgcgtg
     2101 acttcaatcc tatttctgcc tgtgtatgtt tgttagagaa cagttctgac ggccactctg
     2161 aaaggctatt cggtataggc ttcggaccgt acatcatcgc caaccaacac ctatttagaa
     2221 gaaataacgg cgaactgacc atcaaaacca tgcacggtga gttcaaagtt aagaactcca
     2281 ctcagcttca gatgaaacct gttgaaggaa gagatattat agtgattaag atggcaaaag
     2341 actttccgcc atttccacaa aaattgaaat ttcgtcagcc gaccataaaa gatagagttt
     2401 gtatggtaag tactaacttt caacagaaat cagtctcaag tcttgttagc gaaagtagtc
     2461 acattgtgca caaagaggat actagctttt ggcagcactg gataactacg aaagacggtc
     2521 agtgtggtag tccattggta tcaattatag acggaaatat attaggtatc cacagtctaa
     2581 ctcatactac caatggttcc aattatttcg tcgagtttcc tgaaaagttt gtggcgactt
     2641 atctggacgc agccgacgga tggtgcaaaa actggaagtt taacgcagac aaaataagtt
     2701 ggggatcatt caccttagtt gaggatgcac cggaggacgg cattgcataa agccccgaca
     2761 gtaaaaagag accctgacta gccgattcca ggcatcaaat aaaacgaaag gcccagtctt
     2821 tcgactgagc ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac tagagtcaca
     2881 ctggctcacc ttcgggtggg cctttctgcg tttatactcc gggagctgca tgtgtcagag
     2941 gtttaacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     3001 gtttcttaga cgtcaggtgg caggcttact tttcggggaa atgtgcgcgg aacccctatt
     3061 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3121 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     3181 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     3241 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     3301 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     3361 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     3421 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     3481 cttacggatg gcatgacagt aagagaatta tgtagtgctg ccataaccat gagtgataac
     3541 acagcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     3601 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     3661 ataccaaacg acgagcgtga caccacgatg cctgtagcga tggcaacaac gttgcgcaaa
     3721 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     3781 gcggataaag ttgcaggacc acttcttcgc tcagcacttc cagctggttg gtttattgct
     3841 gataaatctg gagccggtga gcgtggatct cgcggtatca tagcagcact ggggccagat
     3901 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     3961 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     4021 caagtttact catatatact ttgaattcaa acttcatttt taatttaaaa ggatctaggt
     4081 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4141 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4201 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4261 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4321 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4381 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4441 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     4501 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4561 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4621 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4681 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatctcg
     4741 tcaggggggc ggagcctatg gaaaaacgcc agcaac
//
