LOCUS       sequence-169949-        6885 bp    DNA     circular SYN 15-APR-2025
DEFINITION  MESA protease chain with rapamycin FKBP ectodomain, 20 extracellular
            linkers, and Tev protease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6885)
  AUTHORS   Hartfield RM, Schwarz KA, Muldoon JJ, Bagheri N, Leonard JN
  TITLE     Multiplexing Engineered Receptors for Multiparametric Evaluation of 
            Environmental Ligands.
  JOURNAL   ACS Synth Biol. 2017 Nov 17;6(11):2042-2055. doi: 
            10.1021/acssynbio.6b00279. Epub 2017 Aug 23.
  PUBMED    28771312
REFERENCE   2  (bases 1 to 6885)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acssynbio.6b00279"; journalName: "ACS Synth Biol"; date: 
            "2017-11-17- 17"; volume: "6"; issue: "11"; pages: "2042-2055"
COMMENT     Sequence Label: Rap-MESA-20-Tev
FEATURES             Location/Qualifiers
     source          1..6885
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        175..554
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        555..758
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     709..729
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     803..822
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        803..821
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(925..927,928..1248)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     misc_feature    1310..1362
                     /label=MCS
                     /note="multiple cloning site"
     CDS             1504..2208
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="ESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKH
                     LFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQR
                     EERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIV
                     GIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPEE
                     PFQPVKEATQLMN"
     polyA_signal    2332..2724
                     /label=beta-globin poly(A)
                     /note="human beta-globin polyadenylation signal"
     rep_origin      3632..4145
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3719..3738)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3929..3950
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4626..4645)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4745..4767
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4805..4823)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4891..4995
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4996..5064,5065..5856)
                     /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(5214..5233)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6027..6615
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6516..6535
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6855..6885,1..99)
                     /label=AAV2 ITR
ORIGIN
        1 gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc
       61 gcagagaggg agtggccaac tccatcacta ggggttcctt gtagttaatg attaacccgc
      121 catgctactt atctacgtag ccatgctcta gggatccggc cagatatacg cgttgacatt
      181 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      241 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      301 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      361 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      421 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      481 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      541 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      601 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      661 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      721 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      781 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      841 gccatcatgt gccgagccat ctctcttagg cgcttgctgc tgctgctgct gcagctgtca
      901 caactcctag ctgtcactca agggatggga gtgcaggtgg aaaccatctc cccaggagac
      961 gggcgcacct tccccaagcg cggccagacc tgcgtggtgc actacaccgg gatgcttgaa
     1021 gatggaaaga aatttgattc ctcccgggac agaaacaagc cctttaagtt tatgctaggc
     1081 aagcaggagg tgatccgagg ctgggaagaa ggggttgccc agatgagtgt gggtcagaga
     1141 gccaaactga ctatatctcc agattatgcc tatggtgcca ctgggcaccc aggcatcatc
     1201 ccaccacatg ccactctcgt cttcgatgtg gagcttctaa aactggaaag tgctggtggt
     1261 agtgctggtg gtagtgctgg tggtagtgct ggtggtagtg ctggtggtcc tcgagctcaa
     1321 gcttcgaatt ctgcagtcga cggtaccgcg ggcccgggat ccaccggatc tagaggcgga
     1381 tctggaggct ctgggggttc aggcggatca ggaggcagtg gcgggtcaac cggtctggtc
     1441 gtggttgctg gagtcctgtt ttgttatggc ttgctagtga cagtggctct ttgtgttgaa
     1501 aacgagagct tgtttaaggg gccgcgtgat tacaacccga tatcgagcac catttgtcat
     1561 ttgacgaatg aatctgatgg gcacacaaca tcgttgtatg gtattggatt tggtcccttc
     1621 atcattacaa acaagcactt gtttagaaga aataatggaa cactgttggt ccaatcacta
     1681 catggtgtat tcaaggtcaa gaacaccacg actttgcaac aacacctcat tgatgggagg
     1741 gacatgataa ttattcgcat gcctaaggat ttcccaccat ttcctcaaaa gctgaaattt
     1801 agagagccac aaagggaaga gcgcatatgt cttgtgacaa ccaacttcca aactaagagc
     1861 atgtctagca tggtgtcaga cactagttgc acattccctt catctgatgg catattctgg
     1921 aagcattgga ttcaaaccaa ggatgggcag tgtggcagtc cattagtatc aactagagat
     1981 gggttcattg ttggtataca ctcagcatcg aatttcacca acacaaacaa ttatttcaca
     2041 agcgtgccga aaaacttcat ggaattgttg acaaatcagg aggcgcagca gtgggttagt
     2101 ggttggcgat taaatgctga ctcagtattg tgggggggcc ataaagtttt catggtgaaa
     2161 cctgaagagc cttttcagcc agttaaggaa gcgactcaac tcatgaatta gtagcggccg
     2221 ccaccgcggt ggagctgttt aaacctcgag caattcaccc caccagtgca ggctgcctat
     2281 cagaaagtgg tggctggtgt ggctaatgcc ctggcccaca agtatcacta agctcgcttt
     2341 cttgctgtcc aatttctatt aaaggttcct ttgttcccta agtccaacta ctaaactggg
     2401 gatattatga agggccttga gcatctggat tctgcctaat aaaaaacatt tattttcatt
     2461 gcaatgatgt atttaaatta tttctgaata ttttactaaa aagggaatgt gggaggtcag
     2521 tgcatttaaa acataaagaa atgaagagct agttcaaacc ttgggaaaat acactatatc
     2581 ttaaactcca tgaaagaagg tgaggctgca aacagtaatg cacattggca acagcccctg
     2641 atgcctatgc cttattcatc cctcagaaaa ggattcaagt agaggcttga tttggaggtt
     2701 aaagttttgc tatgctgtat tttacattac ttattgtttt agctgtcctc atgaatgtct
     2761 tttcactacc catttgctta tcctgcatct ctcagccttg actccactca gttctcttgc
     2821 ttagagatac cacctttccc ctgaagtgtt ccttccatgt tttacggcga gatggtttct
     2881 cctcgcctgg ccactcagcc ttagttgtct ctgttgtctt atagaggtct acttgaagaa
     2941 ggaaaaacag ggggcatggt ttgactgtcc tgtgagccct tcttccctgc ctcccccact
     3001 cacagtgacc cggaatccct cgacatggca gtctagagca tggctacgta gataagtagc
     3061 atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc actccctctc
     3121 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     3181 cccgggcggc ctcagtgagc gagcgagcgc gccagctggc gtaatagcga agaggcccgc
     3241 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggaattc cagacgattg
     3301 agcgtcaaaa tgtaggtatt tccatgagcg tttttcctgt tgcaatggct ggcggtaata
     3361 ttgttctgga tattaccagc aaggccgata gtttgagttc ttctactcag gcaagtgatg
     3421 ttattactaa tcaaagaagt attgcgacaa cggttaattt gcgtgatgga cagactcttt
     3481 tactcggtgg cctcactgat tataaaaaca cttctcagga ttctggcgta ccgttcctgt
     3541 ctaaaatccc tttaatcggc ctcctgttta gctcccgctc tgattctaac gaggaaagca
     3601 cgttatacgt gctcgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     3661 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     3721 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     3781 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     3841 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     3901 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     3961 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     4021 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     4081 acaatttaaa tatttgctta tacaatcttc ctgtttttgg ggcttttctg attatcaacc
     4141 ggggtacata tgattgacat gctagtttta cgattaccgt tcatcgattc tcttgtttgc
     4201 tccagactct caggcaatga cctgatagcc tttgtagaga cctctcaaaa atagctaccc
     4261 tctccggcat gaatttatca gctagaacgg ttgaatatca tattgatggt gatttgactg
     4321 tctccggcct ttctcacccg tttgaatctt tacctacaca ttactcaggc attgcattta
     4381 aaatatatga gggttctaaa aatttttatc cttgcgttga aataaaggct tctcccgcaa
     4441 aagtattaca gggtcataat gtttttggta caaccgattt agctttatgc tctgaggctt
     4501 tattgcttaa ttttgctaat tctttgcctt gcctgtatga tttattggat gttggaattc
     4561 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact
     4621 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     4681 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4741 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     4801 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     4861 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     4921 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     4981 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     5041 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5101 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5161 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5221 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5281 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5341 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5401 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     5461 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     5521 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     5581 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     5641 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     5701 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     5761 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     5821 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     5881 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     5941 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6001 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6061 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6121 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     6181 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6241 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6301 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6361 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6421 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6481 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6541 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6601 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6661 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     6721 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     6781 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     6841 cattaatgca gcagctgcgc gctcgctcgc tcactgaggc cgccc
//
