LOCUS       sequence-487911-        7891 bp    DNA     circular SYN 02-OCT-2025
DEFINITION  Constitutive expression of a MESA NTEVp chain encoding (N to C): 
            TNFR2SS-3xFLAG-TNFR2ECD-mCD28TMD-NTEVp(75S).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7891)
  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 7891)
  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: L4207 TNFR2 NTEVp chain in PolyTX-mNeonGreen
FEATURES             Location/Qualifiers
     source          1..7891
                     /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             1720..1785
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    2971..3195
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             3338..4132
                     /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(3392..3411)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4002..4021
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      4163..4257
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     enhancer        4278..4657
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4658..4861
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4812..4832
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4906..4925
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4906..4924
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4944..4953
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      5205..5214
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    5694..5918
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5964..6392
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6051..6070)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6261..6282
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6415..6611
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6462..6597
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6524..6543
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    6731..6852
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6768..6787)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6822..6841
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6901..6917)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6901..6917)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6914..6936)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6925..6941
                     /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(6949..6955,6956..6973,6974..6979))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6994..7015
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7132..7149)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7303..7891)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7383..7402)
                     /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 accatggctc ccgtggctgt ttgggctgct
     1681 ctggctgttg gactggaact gtgggctgcc gctcacgccg attacaagga tcacgacggc
     1741 gactataagg accacgacat cgattacaaa gacgacgacg acaagctgcc cgctcaggtg
     1801 gcctttacac cttacgctcc agagcctggc agcacctgta gactgagaga gtactacgac
     1861 cagacagccc agatgtgctg ctccaagtgt tctcctggac agcacgccaa ggtgttctgc
     1921 accaagacca gcgacaccgt gtgcgacagc tgcgaggata gcacctacac tcagctgtgg
     1981 aactgggtgc ccgagtgtct gtcttgcggc agcagatgta gcagcgacca ggtggaaaca
     2041 caggcctgca ccagagagca gaaccggatc tgcacatgca gacccggctg gtactgtgcc
     2101 ctgtctaagc aagagggatg cagactgtgc gcccctctga gaaagtgcag acctggcttc
     2161 ggagtggcca gaccaggcac cgagacatct gatgtcgtgt gcaagccttg cgctcccggc
     2221 acctttagca ataccaccag cagcaccgac atctgccggc ctcaccaaat ctgcaacgtg
     2281 gtggccattc ctggcaacgc ctctatggat gccgtgtgta ccagcacaag ccccactcgg
     2341 tctatggctc caggtgctgt tcatctgccc cagcctgtgt ctaccagaag ccagcacaca
     2401 cagcccacac ctgagccttc tacagcccct agcaccagct ttctgctgcc tatgggacct
     2461 tctccaccag ccgaaggcag caccggcgat accggtcttg tcgtcgttgc aggggtgctc
     2521 ttctgttatg gtctgttggt gacagtcgca ctgtgtgtag gaggcggtag cggaggcggc
     2581 gccgagagct tgtttaaggg gccgcgtgat tacaacccga tatcgagcac catttgtcat
     2641 ttgacgaatg aatctgatgg gcacacaaca tcgttgtatg gtattggatt tggtcccttc
     2701 atcattacaa acaagcactt gtttagaaga aataatggaa cactgttggt ccaatcacta
     2761 catggtgtat tcaaggtcaa gaacaccacg actttgcaac aaagcctcat tgatgggagg
     2821 gacatgataa ttattcgcat gcctaaggat ttcccaccat ttcctcaaaa gctgaaattt
     2881 agagagccac aaagggaaga gcgcatatgt cttgtgacaa ccaacttcca aacttaggcg
     2941 gccgcgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     3001 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     3061 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     3121 gggtggggca ggacagcaag ggggaggatt gggaagtcaa tagcaggcat gctggggatg
     3181 cggtgggctc tatgggacgt cggaattgca agttgggccg ccctctggta aggttgggaa
     3241 gccctgcaaa gtaaactgga tggctttctt gccgccaagg atctgatggc gcaggggatc
     3301 aagatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     3361 cgcaggttct ccggcggctt gggtggagag gctattcggc tatgactggg cacaacagac
     3421 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg cagggtcgcc cggttctttt
     3481 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc
     3541 gtggctggcc acgacgggcg ttccttgcgc ggctgtgctc gacgttgtca ctgaagcggg
     3601 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     3661 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     3721 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     3781 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     3841 cgaactgttc gccaggctca aggcgcgtat gcccgacggc gaggatctcg tcgtgaccca
     3901 cggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     3961 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     4021 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     4081 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     4141 ctggggttcg agaattggct tggactcctg ttgatagatc cagtaatgac ctcagaactc
     4201 catctggatt tgttcagaac gctcggttgc cgccgggcgt tttttattgg tgagaatcca
     4261 agcactagcc atgaccagac attgattatt gactagttat taatagtaat caattacggg
     4321 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     4381 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     4441 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     4501 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
     4561 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
     4621 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
     4681 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
     4741 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
     4801 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
     4861 tctctggcta actagagaac ccactgctta ctggcttatc gaaattaata cgactcacta
     4921 tagggagtcc caagctgact agcgccacca tggtttccaa gggcgaagag gacaatatgg
     4981 ccagcctgcc tgccacacac gagctgcaca tctttggcag catcaacggc gtggacttcg
     5041 acatggtcgg acaaggcacc ggcaatccca acgacggcta cgaggaactg aacctgaagt
     5101 ccacaaaggg cgacctccag ttcagcccct ggattctggt gcctcacatc ggctacggct
     5161 tccaccagta cctgccttat cctgacggca tgagcccttt ccaggccgcc atggttgatg
     5221 gctctggcta ccaggtccac cggaccatgc agtttgagga tggcgcttcc ctgaccgtga
     5281 actacagata cacctacgag ggcagccaca tcaaaggcga ggcccaagtg aaaggcacag
     5341 gcttccctgc tgatggcccc gtgatgacca attctctgac agccgccgac tggtgcagaa
     5401 gcaagaaaac ataccccaac gacaagacca tcatcagcac cttcaagtgg tcctacacca
     5461 ccggcaacgg caagcggtac agaagcaccg ccagaaccac ctacaccttc gccaaaccta
     5521 tggccgccaa ctacctgaag aaccagccta tgtacgtgtt ccgcaagacc gagctgaagc
     5581 actccaagac agaactgaac ttcaaagagt ggcagaaagc cttcaccgac gtgatgggca
     5641 tggacgaact ctacaaatga gcggccaagt ttaaacccgc tgatcagcct cgactgtgcc
     5701 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     5761 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     5821 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaagt
     5881 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     5941 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     6001 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     6061 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     6121 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     6181 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     6241 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     6301 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     6361 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggc
     6421 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     6481 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     6541 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     6601 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggaggttgg gcttcggaat
     6661 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     6721 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     6781 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     6841 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     6901 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     6961 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     7021 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     7081 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     7141 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     7201 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     7261 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     7321 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     7381 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     7441 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     7501 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     7561 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     7621 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     7681 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     7741 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     7801 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     7861 gcagattacg cgcagaaaaa aaggatctca a
//
