LOCUS       sequence-487910-        8290 bp    DNA     circular SYN 02-OCT-2025
DEFINITION  Constitutive expression of a MESA CTEVp chain encoding (N to C): 
            TNFR1SS-3xFLAG-TNFR1ECD(dMMP)-TNFR1TMD-CTEVp(75S)-PRS(M)-VP64-ZF6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8290)
  AUTHORS   Edelstein HI, Cosio A, Ezekiel ML, Corcoran WK, Morris AH, Leonard 
            JN
  TITLE     Conversion of natural cytokine receptors into orthogonal synthetic 
            biosensors.
  JOURNAL   Nat Chem Biol. 2025 Aug 22:10.1038/s41589-025-01986-1. doi: 
            10.1038/s41589-025-01986-1.
  PUBMED    40846994
REFERENCE   2  (bases 1 to 8290)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 2, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Chem 
            Biol. 2025 Aug 22:10.1038/s41589-025-01986-1. doi: 
            10.1038/s41589-025-01986-1."
COMMENT     Sequence Label: L4342 TNFR1 CTEVp chain in PolyTX-mTagBFP2
FEATURES             Location/Qualifiers
     source          1..8290
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        359..1541
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          590..1532
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1489..1509
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1543..1567
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1648..1657
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1741..1806
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2788..2808
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQM"
     CDS             2827..2847
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2854..3003
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     polyA_signal    3367..3591
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             3734..4528
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3788..3807)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4398..4417
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      4559..4653
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     enhancer        4674..5053
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5054..5257
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5208..5228
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5302..5321
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5302..5320
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      5340..5349
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    6093..6317
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6363..6791
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6450..6469)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6660..6681
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6814..7010
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6861..6996
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6923..6942
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    7130..7251
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7167..7186)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7221..7240
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7300..7316)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7300..7316)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7313..7335)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7324..7340
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(7348..7354,7355..7372,7373..7378))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7393..7414
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7531..7548)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7702..8290)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7782..7801)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag cgatcgaata
      181 ttagatctga gacccaattg acgcgtgagt aattcataca aaaggactcg cccctgcctt
      241 ggggaatccc agggaccgtc gttaaactcc cactaacgta gaacccagag atcgctgcgt
      301 tcccgccccc tcacccgccc gctctcgtca tcactgaggt ggagaagagc atgcgtgagg
      361 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
      421 aggggtcggc aattgaaccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
      481 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
      541 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt
      601 gtgtggttcc cgcgggcctg gcctctttac gggttatggc ccttgcgtgc cttgaattac
      661 ttccacgccc ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt
      721 gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct
      781 ggcttgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg
      841 ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct
      901 ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg
      961 ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc
     1021 gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc
     1081 ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac
     1141 cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga
     1201 ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc
     1261 cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg
     1321 attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga
     1381 tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt
     1441 aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga
     1501 cagtggttca aagttttttt cttccatttc aggtgtcgtg agagctcgtt tagtgaaccg
     1561 tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagag aacgggaccg
     1621 atccaaattc tcgaggctag ctgtacagcc accatgggcc tgagcacagt gcctgatttg
     1681 ctgctgcctc tggtgctgct ggaactgctc gtgggcatct atcctagcgg cgtgatcggc
     1741 gactacaagg accacgacgg cgattataag gatcatgaca tcgattacaa ggacgatgac
     1801 gacaagctgg tgccccacct gggcgataga gaaaagcggg atagcgtgtg ccctcagggc
     1861 aagtacattc accctcagaa caacagcatc tgctgcacca agtgccacaa gggcacctac
     1921 ctgtataacg actgccctgg accaggccag gacaccgatt gcagagagtg tgaaagcggc
     1981 agcttcaccg ccagcgagaa ccacctgaga cactgcctgt cctgctccaa gtgcagaaaa
     2041 gagatgggcc aagtcgagat cagcagctgc accgtggaca gagataccgt gtgcggctgc
     2101 cggaagaacc agtacagaca ctattggagc gagaatctgt tccagtgctt caactgctcc
     2161 ctgtgcctga acggcaccgt gcatctgagc tgccaagaga agcagaacac cgtgtgtacc
     2221 tgccacgccg gattctttct gcgcgagaac gagtgcgtgt cctgtagcaa ctgcaagaaa
     2281 agcctggaat gcaccaagct gtgcctgcct cagggcacag aggatagcgg caccacagtg
     2341 ctgctccctc tcgtgatctt cttcggcctg tgtctgctga gcctgctgtt catcggcctg
     2401 atgtatagat accagggcgc caagagcatg tctagcatgg tgtcagacac tagttgcaca
     2461 ttcccttcat ctgatggcat attctggaag cattggattc aaaccaagga tgggcagtgt
     2521 ggcagtccat tagtatcaac tagagatggg ttcattgttg gtatacactc agcatcgaat
     2581 ttcaccaaca caaacaatta tttcacaagc gtgccgaaaa acttcatgga attgaagaca
     2641 aatcaggagg cgcagcagtg ggttagtggt tggcgattaa atgctgactc agtattgtgg
     2701 gggggccata aagttttcat ggtgaaacct gaagagcctt ttcagccagt taaggaagcg
     2761 actcaactca tgaatcttaa gggctcggag aacctgtatt ttcagatggg gggaggcagc
     2821 gggggcccta agaaaaagcg caaagtctcc ggagacgctc tggatgactt tgatctggac
     2881 atgcttggtt ccgatgcctt ggacgatttt gacctcgata tgctgggctc cgacgctctc
     2941 gacgatttcg acctcgacat gctgggtagc gatgcactgg atgactttga tctggatatg
     3001 ctcggatccg gtggtggcgg gtctggagga ggaggtagtg gtggaggtgg ctctggtacc
     3061 gctagacccg gcgaaagacc tttccagtgc cggatctgca tgaggaactt cagcaagggc
     3121 gagagactcg tgcggcacac cagaacacac acaggcgaga agcccttcca gtgtagaatc
     3181 tgtatgcgca acttcagccg gatggacaac ctgagcaccc acctgagaac ccataccggg
     3241 gagaagccat ttcaatgccg catctgtatg agaaattttt cccggaagga cgccctgaac
     3301 cggcacctga aaacacacct gagaggcagc tgagcggccg cgtttaaacc cgctgatcag
     3361 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     3421 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     3481 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     3541 aggattggga agtcaatagc aggcatgctg gggatgcggt gggctctatg ggacgtcgga
     3601 attgcaagtt gggccgccct ctggtaaggt tgggaagccc tgcaaagtaa actggatggc
     3661 tttcttgccg ccaaggatct gatggcgcag gggatcaaga tctgatcaag agacaggatg
     3721 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg cggcttgggt
     3781 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     3841 gttccggctg tcagcgcagg gtcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     3901 cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     3961 ttgcgcggct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     4021 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     4081 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     4141 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     4201 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     4261 gcgtatgccc gacggcgagg atctcgtcgt gacccacggc gatgcctgct tgccgaatat
     4321 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     4381 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     4441 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     4501 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgagaa ttggcttgga
     4561 ctcctgttga tagatccagt aatgacctca gaactccatc tggatttgtt cagaacgctc
     4621 ggttgccgcc gggcgttttt tattggtgag aatccaagca ctagccatga ccagacattg
     4681 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
     4741 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     4801 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     4861 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     4921 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     4981 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     5041 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
     5101 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
     5161 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
     5221 taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac
     5281 tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagtcccaag ctgactagcg
     5341 ccaccatggt ttccaagggc gaagaactga tcaaagaaaa catgcacatg aagctctaca
     5401 tggaaggcac cgtggacaac caccacttca agtgtaccag cgaaggcgag ggcaagcctt
     5461 acgagggcac ccagaccatg agaatcaagg tggtggaagg cggccctctg cctttcgcct
     5521 ttgatatcct ggccaccagc tttctgtacg gcagcaagac cttcatcaat cacacccagg
     5581 gcatccccga tttcttcaag cagagcttcc ccgagggctt cacctgggag agagtgacca
     5641 catacgagga tggcggcgtg ctgacagcca cacaggatac aagcctccag gacggctgcc
     5701 tgatctacaa cgtgaagatc cggggcgtga acttcaccag caacggcccc gtgatgcaga
     5761 agaaaaccct tggctgggaa gccttcaccg agacactgta tcctgccgat ggcggcctgg
     5821 aaggcagaaa cgatatggcc ctgaagctcg tcggcggctc tcacctgatt gccaatgcca
     5881 agaccaccta cagaagcaag aagcccgcca agaacctgaa gatgcccggc gtgtactacg
     5941 tggactaccg gctggaaaga atcaaagagg ccaacaacga gacatacgtg gaacagcacg
     6001 aggtggccgt ggccagatac tgtgatctgc cttctaagct gggccacaag ctgaactgag
     6061 cggccaagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     6121 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     6181 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     6241 tggggtgggg caggacagca agggggagga ttgggaagtc aatagcaggc atgctgggga
     6301 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     6361 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     6421 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     6481 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     6541 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     6601 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     6661 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     6721 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     6781 taacgcgaat taattctgtg gaatgtgtgt cagttaggct caattagtca gcaaccatag
     6841 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     6901 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     6961 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     7021 gagcttgtat atccattttc ggaggttggg cttcggaatc gttttccggg acgccggctg
     7081 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     7141 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     7201 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     7261 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     7321 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     7381 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     7441 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     7501 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     7561 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     7621 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     7681 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     7741 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     7801 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     7861 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     7921 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     7981 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     8041 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     8101 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     8161 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     8221 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     8281 aggatctcaa
//
