LOCUS       sequence-176716-        8256 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MESA target chain with V1-MESA ectodomain, 45 extracellular linkers,
            a flag tag, M cleavage sequence, and tTA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8256)
  AUTHORS   Schwarz KA, Daringer NM, Dolberg TB, Leonard JN
  TITLE     Rewiring human cellular input-output using modular extracellular 
            sensors.
  JOURNAL   Nat Chem Biol. 2016 Dec 12. doi: 10.1038/nchembio.2253.
  PUBMED    27941759
REFERENCE   2  (bases 1 to 8256)
  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: "Nat Chem 
            Biol. 2016 Dec 12. doi: 10.1038/nchembio.2253."
COMMENT     Sequence Label: V1-MESA-45F-M-tTA
FEATURES             Location/Qualifiers
     source          1..8256
                     /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             1807..1827
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQM"
     CDS             1825..2448
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     CDS             join(1828..2448,2449..2832)
                     /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(1901..1920)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             join(2863..2865,2866..2868,2869..3582)
                     /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(2908..2929)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    3703..4095
                     /label=beta-globin poly(A)
                     /note="human beta-globin polyadenylation signal"
     rep_origin      5003..5516
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5090..5109)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5300..5321
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5997..6016)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6116..6138
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6176..6194)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6262..6366
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6367..6435,6436..7227)
                     /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(6585..6604)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7398..7986
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7887..7906
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(8226..8256,1..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 gccatcatgg cttggaccag cttaattctt agcttgcttg ccctgtgcag cggtgcatca
      901 tctgacatac agatgactca gtcacctagc agtctgtctg cttcagtcgg tgatagggta
      961 actatcacct gtcgggcatc tcaggatgtg tccacggcag ttgcctggta ccagcagaag
     1021 cccggcaagg cgcctaagtt gcttatctat tcagcttcat ttttgtatag tggggtgcct
     1081 agccggttta gcggaagtgg ttctggcaca gacttcactt tgacaatcag ttccctccaa
     1141 ccggaggatt tcgctaccta ctactgtcag caggggtacg gaaacccctt tacgtttgga
     1201 cagggcacta aagtggagat taagagggga ggcggaggct ccggtggggg aggctctggt
     1261 ggtggaggta gcgaggttca gctggtggaa agtggtggcg gactagtaca gccaggcggc
     1321 tccctgcggc tgagttgcgc cgcgagtggc tttactatat ctgactattg gatccattgg
     1381 gtcaggcaag cccctgggaa aggattggag tgggttgctg gtattacccc cgcaggtggg
     1441 tatacttatt atgcagatag cgtgaaaggg aggttcacca tctctgcaga cacaagcaag
     1501 aacacggcct acctacagat gaactctctg agggcagagg acactgccgt ctactattgc
     1561 gcccgatttg tgttttttct accctacgcc atggactatt ggggacaggg gacattggta
     1621 ggcgccggga caggagggtc tgggggtggc actggcggat ctggaggaga ctacaaggac
     1681 gatgacgaca acggcggatc gggggggtct ggtggtagtg gcggttcggg gggaggtacc
     1741 ggtctggtcg tggttgctgg agtcctgttt tgttatggct tgctagtgac agtggctctt
     1801 tgtgttgaaa acctgtattt tcagatgatg tctagattag ataaaagtaa agtgattaac
     1861 agcgcattag agctgcttaa tgaggtcgga atcgaaggtt taacaacccg taaactcgcc
     1921 cagaagctag gtgtagagca gcctacattg tattggcatg taaaaaataa gcgggctttg
     1981 ctcgacgcct tagccattga gatgttagat aggcaccata ctcacttttg ccctttagaa
     2041 ggggaaagct ggcaagattt tttacgtaat aacgctaaaa gttttagatg tgctttacta
     2101 agtcatcgcg atggagcaaa agtacattta ggtacacggc ctacagaaaa acagtatgaa
     2161 actctcgaaa atcaattagc ctttttatgc caacaaggtt tttcactaga gaatgcatta
     2221 tatgcactca gcgctgtggg gcattttact ttaggttgcg tattggaaga tcaagagcat
     2281 caagtcgcta aagaagaaag ggaaacacct actactgata gtatgccgcc attattacga
     2341 caagctatcg aattatttga tcaccaaggt gcagagccag ccttcttatt cggccttgaa
     2401 ttgatcatat gcggattaga aaaacaactt aaatgtgaaa gtgggtccgc gtacagccgc
     2461 gcgcgtacga aaaacaatta cgggtctacc atcgagggcc tgctcgatct cccggacgac
     2521 gacgcccccg aagaggcggg gctggcggct ccgcgcctgt cctttctccc cgcgggacac
     2581 acgcgcagac tgtcgacggc ccccccgacc gatgtcagcc tgggggacga gctccactta
     2641 gacggcgagg acgtggcgat ggcgcatgcc gacgcgctag acgatttcga tctggacatg
     2701 ttgggggacg gggattcccc gggtccggga tttacccccc acgactccgc cccctacggc
     2761 gctctggata tggccgactt cgagtttgag cagatgttta ccgatgccct tggaattgac
     2821 gagtacggtg ggggtaccgg cggaggctcc ggtggtggct ctatggtgag caagggcgag
     2881 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     2941 aagttcagcg tgaggggcga gggcgagggc gatgccacca acggcaagct gaccctgaag
     3001 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgagc
     3061 cacggcgtgc agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag
     3121 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcacc
     3181 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tagtgaaccg catcgagctg
     3241 aagggcgtcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaacttc
     3301 aacagccaca acatctatat catggccgtc aagcagaaga acggcatcaa ggtgaacttc
     3361 aagatccgcc acaacgtgga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     3421 acccccatcg gcgacggccc cgtgctgctg cccgacagcc actacctgag cacccagtcc
     3481 gtgctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttccgcacc
     3541 gccgccggga tcactctcgg catggacgag ctgtacaagt aatagcggcc gccaccgcgg
     3601 tggagctgtt taaacctcga gcaattcacc ccaccagtgc aggctgccta tcagaaagtg
     3661 gtggctggtg tggctaatgc cctggcccac aagtatcact aagctcgctt tcttgctgtc
     3721 caatttctat taaaggttcc tttgttccct aagtccaact actaaactgg ggatattatg
     3781 aagggccttg agcatctgga ttctgcctaa taaaaaacat ttattttcat tgcaatgatg
     3841 tatttaaatt atttctgaat attttactaa aaagggaatg tgggaggtca gtgcatttaa
     3901 aacataaaga aatgaagagc tagttcaaac cttgggaaaa tacactatat cttaaactcc
     3961 atgaaagaag gtgaggctgc aaacagtaat gcacattggc aacagcccct gatgcctatg
     4021 ccttattcat ccctcagaaa aggattcaag tagaggcttg atttggaggt taaagttttg
     4081 ctatgctgta ttttacatta cttattgttt tagctgtcct catgaatgtc ttttcactac
     4141 ccatttgctt atcctgcatc tctcagcctt gactccactc agttctcttg cttagagata
     4201 ccacctttcc cctgaagtgt tccttccatg ttttacggcg agatggtttc tcctcgcctg
     4261 gccactcagc cttagttgtc tctgttgtct tatagaggtc tacttgaaga aggaaaaaca
     4321 gggggcatgg tttgactgtc ctgtgagccc ttcttccctg cctcccccac tcacagtgac
     4381 ccggaatccc tcgacatggc agtctagagc atggctacgt agataagtag catggcgggt
     4441 taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     4501 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
     4561 cctcagtgag cgagcgagcg cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     4621 ccttcccaac agttgcgcag cctgaatggc gaatggaatt ccagacgatt gagcgtcaaa
     4681 atgtaggtat ttccatgagc gtttttcctg ttgcaatggc tggcggtaat attgttctgg
     4741 atattaccag caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta
     4801 atcaaagaag tattgcgaca acggttaatt tgcgtgatgg acagactctt ttactcggtg
     4861 gcctcactga ttataaaaac acttctcagg attctggcgt accgttcctg tctaaaatcc
     4921 ctttaatcgg cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg
     4981 tgctcgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     5041 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     5101 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     5161 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     5221 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     5281 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     5341 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     5401 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttaa
     5461 atatttgctt atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat
     5521 atgattgaca tgctagtttt acgattaccg ttcatcgatt ctcttgtttg ctccagactc
     5581 tcaggcaatg acctgatagc ctttgtagag acctctcaaa aatagctacc ctctccggca
     5641 tgaatttatc agctagaacg gttgaatatc atattgatgg tgatttgact gtctccggcc
     5701 tttctcaccc gtttgaatct ttacctacac attactcagg cattgcattt aaaatatatg
     5761 agggttctaa aaatttttat ccttgcgttg aaataaaggc ttctcccgca aaagtattac
     5821 agggtcataa tgtttttggt acaaccgatt tagctttatg ctctgaggct ttattgctta
     5881 attttgctaa ttctttgcct tgcctgtatg atttattgga tgttggaatt cctgatgcgg
     5941 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca
     6001 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     6061 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     6121 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     6181 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     6241 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     6301 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     6361 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     6421 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     6481 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     6541 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     6601 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     6661 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     6721 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     6781 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     6841 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     6901 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     6961 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     7021 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     7081 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     7141 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     7201 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     7261 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     7321 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     7381 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     7441 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     7501 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     7561 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     7621 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     7681 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     7741 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     7801 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     7861 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     7921 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     7981 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     8041 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     8101 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     8161 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     8221 agcagctgcg cgctcgctcg ctcactgagg ccgccc
//
