LOCUS       sequence-487905-        7729 bp    DNA     circular SYN 02-OCT-2025
DEFINITION  Constitutive expression of a MESA NTEVp chain encoding (N to C): 
            TNFR1SS-3xFLAG-TNFR1ECD(dMMP)-TNFR1TMD-NTEVp(75S).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7729)
  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 7729)
  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: L4341 TNFR1 NTEVp chain (deleted MMP site) in
            ...
FEATURES             Location/Qualifiers
     source          1..7729
                     /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"
     polyA_signal    2809..3033
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             3176..3970
                     /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(3230..3249)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3840..3859
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      4001..4095
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     enhancer        4116..4495
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4496..4699
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4650..4670
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4744..4763
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4744..4762
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4782..4791
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      5043..5052
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    5532..5756
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5802..6230
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5889..5908)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6099..6120
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6253..6449
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6300..6435
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6362..6381
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    6569..6690
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6606..6625)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6660..6679
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6739..6755)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6739..6755)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6752..6774)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6763..6779
                     /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(6787..6793,6794..6811,6812..6817))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6832..6853
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6970..6987)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7141..7729)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7221..7240)
                     /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 cgagagcttg tttaaggggc cgcgtgatta caacccgata
     2461 tcgagcacca tttgtcattt gacgaatgaa tctgatgggc acacaacatc gttgtatggt
     2521 attggatttg gtcccttcat cattacaaac aagcacttgt ttagaagaaa taatggaaca
     2581 ctgttggtcc aatcactaca tggtgtattc aaggtcaaga acaccacgac tttgcaacaa
     2641 agcctcattg atgggaggga catgataatt attcgcatgc ctaaggattt cccaccattt
     2701 cctcaaaagc tgaaatttag agagccacaa agggaagagc gcatatgtct tgtgacaacc
     2761 aacttccaaa cttaggcggc cgcgtttaaa cccgctgatc agcctcgact gtgccttcta
     2821 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     2881 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     2941 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagtcaata
     3001 gcaggcatgc tggggatgcg gtgggctcta tgggacgtcg gaattgcaag ttgggccgcc
     3061 ctctggtaag gttgggaagc cctgcaaagt aaactggatg gctttcttgc cgccaaggat
     3121 ctgatggcgc aggggatcaa gatctgatca agagacagga tgaggatcgt ttcgcatgat
     3181 tgaacaagat ggattgcacg caggttctcc ggcggcttgg gtggagaggc tattcggcta
     3241 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     3301 gggtcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga
     3361 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcgg ctgtgctcga
     3421 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     3481 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     3541 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     3601 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     3661 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgtatgc ccgacggcga
     3721 ggatctcgtc gtgacccacg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     3781 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     3841 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     3901 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     3961 gttcttctga gcgggactct ggggttcgag aattggcttg gactcctgtt gatagatcca
     4021 gtaatgacct cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt
     4081 tttattggtg agaatccaag cactagccat gaccagacat tgattattga ctagttatta
     4141 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     4201 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     4261 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     4321 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     4381 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     4441 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
     4501 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
     4561 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
     4621 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
     4681 ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga
     4741 aattaatacg actcactata gggagtccca agctgactag cgccaccatg gtttccaagg
     4801 gcgaagagga caatatggcc agcctgcctg ccacacacga gctgcacatc tttggcagca
     4861 tcaacggcgt ggacttcgac atggtcggac aaggcaccgg caatcccaac gacggctacg
     4921 aggaactgaa cctgaagtcc acaaagggcg acctccagtt cagcccctgg attctggtgc
     4981 ctcacatcgg ctacggcttc caccagtacc tgccttatcc tgacggcatg agccctttcc
     5041 aggccgccat ggttgatggc tctggctacc aggtccaccg gaccatgcag tttgaggatg
     5101 gcgcttccct gaccgtgaac tacagataca cctacgaggg cagccacatc aaaggcgagg
     5161 cccaagtgaa aggcacaggc ttccctgctg atggccccgt gatgaccaat tctctgacag
     5221 ccgccgactg gtgcagaagc aagaaaacat accccaacga caagaccatc atcagcacct
     5281 tcaagtggtc ctacaccacc ggcaacggca agcggtacag aagcaccgcc agaaccacct
     5341 acaccttcgc caaacctatg gccgccaact acctgaagaa ccagcctatg tacgtgttcc
     5401 gcaagaccga gctgaagcac tccaagacag aactgaactt caaagagtgg cagaaagcct
     5461 tcaccgacgt gatgggcatg gacgaactct acaaatgagc ggccaagttt aaacccgctg
     5521 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     5581 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     5641 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     5701 gggggaggat tgggaagtca atagcaggca tgctggggat gcggtgggct ctatggcttc
     5761 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     5821 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     5881 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     5941 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     6001 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     6061 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     6121 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     6181 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     6241 aatgtgtgtc agttaggctc aattagtcag caaccatagt cccgccccta actccgccca
     6301 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     6361 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag
     6421 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg
     6481 gaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     6541 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     6601 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     6661 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     6721 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     6781 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     6841 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     6901 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     6961 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     7021 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     7081 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     7141 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     7201 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     7261 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     7321 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     7381 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     7441 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     7501 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     7561 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     7621 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     7681 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaa
//
