LOCUS       sequence-99612-e        7737 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MESA target chain with Rapamycin FRB ectodomain, no linker domain, G
            cleavage sequence, and tTA-BFP fusion .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7737)
  AUTHORS   Daringer NM, Dudek RM, Schwarz KA, Leonard JN
  TITLE     Modular Extracellular Sensor Architecture for Engineering Mammalian 
            Cell-based Devices.
  JOURNAL   ACS Synth Biol. 2014 Mar 11.
  PUBMED    24611683
REFERENCE   2  (bases 1 to 7737)
  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: "ACS Synth 
            Biol. 2014 Mar 11."
COMMENT     Sequence Label: FRB_LD 0_CS G_tTA-BFP
FEATURES             Location/Qualifiers
     source          1..7737
                     /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             1288..1308
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1309..1929,1930..2313)
                     /codon_start=1
                     /product="tetracycline-controlled transactivator, 
                     comprising a fusion of the tetracycline repressor TetR with
                     the C-terminal activation domain of herpes simplex virus 
                     VP16"
                     /label=tTA
                     /note="In the Tet-Off(R) system, tTA binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTI
                     EGLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHA
                     DALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     primer_bind     complement(1382..1401)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             join(2344..2346,2347..2349,2350..3063)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(2389..2410)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    3184..3576
                     /label=beta-globin poly(A)
                     /note="human beta-globin polyadenylation signal"
     rep_origin      4484..4997
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4571..4590)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4781..4802
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5478..5497)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5597..5619
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5657..5675)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5743..5847
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5848..5916,5917..6708)
                     /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(6066..6085)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6879..7467
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7368..7387
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(7707..7737,1..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 agggatgctc gagatgtggc atgaaggcct ggaagaggca
      961 tctcgtttgt actttgggga aaggaacgtg aaaggcatgt ttgaggtgct ggagcccttg
     1021 catgctatga tggaacgggg cccccagact ctgaaggaaa catcctttaa tcaggcctat
     1081 ggtcgagatt taatggaggc ccaagagtgg tgcaggaagt acatgaaatc agggaatgtc
     1141 aaggacctcc tccaagcctg ggacctctat tatcatgtgt tccgacgaat ctcaaagact
     1201 agaaccggta agcttttttg ggcactggtc gtggttgctg gagtcctgtt ttgttatggc
     1261 ttgctagtga cagtggctct ttgtgttgaa aacctgtatt ttcagggtat gtctagatta
     1321 gataaaagta aagtgattaa cagcgcatta gagctgctta atgaggtcgg aatcgaaggt
     1381 ttaacaaccc gtaaactcgc ccagaagcta ggtgtagagc agcctacatt gtattggcat
     1441 gtaaaaaata agcgggcttt gctcgacgcc ttagccattg agatgttaga taggcaccat
     1501 actcactttt gccctttaga aggggaaagc tggcaagatt ttttacgtaa taacgctaaa
     1561 agttttagat gtgctttact aagtcatcgc gatggagcaa aagtacattt aggtacacgg
     1621 cctacagaaa aacagtatga aactctcgaa aatcaattag cctttttatg ccaacaaggt
     1681 ttttcactag agaatgcatt atatgcactc agcgctgtgg ggcattttac tttaggttgc
     1741 gtattggaag atcaagagca tcaagtcgct aaagaagaaa gggaaacacc tactactgat
     1801 agtatgccgc cattattacg acaagctatc gaattatttg atcaccaagg tgcagagcca
     1861 gccttcttat tcggccttga attgatcata tgcggattag aaaaacaact taaatgtgaa
     1921 agtgggtccg cgtacagccg cgcgcgtacg aaaaacaatt acgggtctac catcgagggc
     1981 ctgctcgatc tcccggacga cgacgccccc gaagaggcgg ggctggcggc tccgcgcctg
     2041 tcctttctcc ccgcgggaca cacgcgcaga ctgtcgacgg cccccccgac cgatgtcagc
     2101 ctgggggacg agctccactt agacggcgag gacgtggcga tggcgcatgc cgacgcgcta
     2161 gacgatttcg atctggacat gttgggggac ggggattccc cgggtccggg atttaccccc
     2221 cacgactccg ccccctacgg cgctctggat atggccgact tcgagtttga gcagatgttt
     2281 accgatgccc ttggaattga cgagtacggt ggaggtaccg gcggaggctc cggtggtggc
     2341 tctatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     2401 gacggcgacg taaacggcca caagttcagc gtgaggggcg agggcgaggg cgatgccacc
     2461 aacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     2521 accctcgtga ccaccctgag ccacggcgtg cagtgcttcg cccgctaccc cgaccacatg
     2581 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     2641 ttcttcaagg acgacggcac ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     2701 ctagtgaacc gcatcgagct gaagggcgtc gacttcaagg aggacggcaa catcctgggg
     2761 cacaagctgg agtacaactt caacagccac aacatctata tcatggccgt caagcagaag
     2821 aacggcatca aggtgaactt caagatccgc cacaacgtgg aggacggcag cgtgcagctc
     2881 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacagc
     2941 cactacctga gcacccagtc cgtgctgagc aaagacccca acgagaagcg cgatcacatg
     3001 gtcctgctgg agttccgcac cgccgccggg atcactctcg gcatggacga gctgtacaag
     3061 taatagcggc cgccaccgcg gtggagctgt ttaaacctcg agcaattcac cccaccagtg
     3121 caggctgcct atcagaaagt ggtggctggt gtggctaatg ccctggccca caagtatcac
     3181 taagctcgct ttcttgctgt ccaatttcta ttaaaggttc ctttgttccc taagtccaac
     3241 tactaaactg gggatattat gaagggcctt gagcatctgg attctgccta ataaaaaaca
     3301 tttattttca ttgcaatgat gtatttaaat tatttctgaa tattttacta aaaagggaat
     3361 gtgggaggtc agtgcattta aaacataaag aaatgaagag ctagttcaaa ccttgggaaa
     3421 atacactata tcttaaactc catgaaagaa ggtgaggctg caaacagtaa tgcacattgg
     3481 caacagcccc tgatgcctat gccttattca tccctcagaa aaggattcaa gtagaggctt
     3541 gatttggagg ttaaagtttt gctatgctgt attttacatt acttattgtt ttagctgtcc
     3601 tcatgaatgt cttttcacta cccatttgct tatcctgcat ctctcagcct tgactccact
     3661 cagttctctt gcttagagat accacctttc ccctgaagtg ttccttccat gttttacggc
     3721 gagatggttt ctcctcgcct ggccactcag ccttagttgt ctctgttgtc ttatagaggt
     3781 ctacttgaag aaggaaaaac agggggcatg gtttgactgt cctgtgagcc cttcttccct
     3841 gcctccccca ctcacagtga cccggaatcc ctcgacatgg cagtctagag catggctacg
     3901 tagataagta gcatggcggg ttaatcatta actacaagga acccctagtg atggagttgg
     3961 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     4021 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgccagctg gcgtaatagc
     4081 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggaat
     4141 tccagacgat tgagcgtcaa aatgtaggta tttccatgag cgtttttcct gttgcaatgg
     4201 ctggcggtaa tattgttctg gatattacca gcaaggccga tagtttgagt tcttctactc
     4261 aggcaagtga tgttattact aatcaaagaa gtattgcgac aacggttaat ttgcgtgatg
     4321 gacagactct tttactcggt ggcctcactg attataaaaa cacttctcag gattctggcg
     4381 taccgttcct gtctaaaatc cctttaatcg gcctcctgtt tagctcccgc tctgattcta
     4441 acgaggaaag cacgttatac gtgctcgtca aagcaaccat agtacgcgcc ctgtagcggc
     4501 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     4561 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     4621 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     4681 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     4741 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     4801 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     4861 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     4921 atattaacgt ttacaattta aatatttgct tatacaatct tcctgttttt ggggcttttc
     4981 tgattatcaa ccggggtaca tatgattgac atgctagttt tacgattacc gttcatcgat
     5041 tctcttgttt gctccagact ctcaggcaat gacctgatag cctttgtaga gacctctcaa
     5101 aaatagctac cctctccggc atgaatttat cagctagaac ggttgaatat catattgatg
     5161 gtgatttgac tgtctccggc ctttctcacc cgtttgaatc tttacctaca cattactcag
     5221 gcattgcatt taaaatatat gagggttcta aaaattttta tccttgcgtt gaaataaagg
     5281 cttctcccgc aaaagtatta cagggtcata atgtttttgg tacaaccgat ttagctttat
     5341 gctctgaggc tttattgctt aattttgcta attctttgcc ttgcctgtat gatttattgg
     5401 atgttggaat tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     5461 atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     5521 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     5581 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     5641 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     5701 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5761 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5821 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     5881 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     5941 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     6001 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     6061 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     6121 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     6181 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     6241 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     6301 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     6361 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6421 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6481 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     6541 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     6601 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     6661 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     6721 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6781 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6841 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6901 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6961 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     7021 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     7081 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     7141 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     7201 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     7261 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     7321 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     7381 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     7441 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     7501 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     7561 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     7621 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     7681 cgttggccga ttcattaatg cagcagctgc gcgctcgctc gctcactgag gccgccc
//
