LOCUS       sequence-487902-        7792 bp    DNA     circular SYN 02-OCT-2025
DEFINITION  Constitutive expression of a MESA NTEVp chain encoding (N to C): 
            hCD8aSS-3xFLAG-IL10RbECD-IL10RbTMD-NTEVp(75S).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7792)
  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 7792)
  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: L4312 IL-10Rb NTEVp chain (human CD8a SS) in
            P...
FEATURES             Location/Qualifiers
     source          1..7792
                     /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             1717..1782
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    2872..3096
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             3239..4033
                     /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(3293..3312)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3903..3922
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      4064..4158
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     enhancer        4179..4558
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4559..4762
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4713..4733
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4807..4826
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4807..4825
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4845..4854
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      5106..5115
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    5595..5819
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5865..6293
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5952..5971)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6162..6183
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6316..6512
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6363..6498
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6425..6444
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    6632..6753
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6669..6688)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6723..6742
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6802..6818)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6802..6818)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6815..6837)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6826..6842
                     /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(6850..6856,6857..6874,6875..6880))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6895..6916
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7033..7050)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7204..7792)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7284..7303)
                     /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 accatggccc tgcctgtgac agccctgctg
     1681 ctgcctctgg ctctgctgct gcacgccgct agacccgact acaaggacca cgacggcgat
     1741 tataaggatc atgacatcga ttacaaggac gatgacgaca agatggtgcc tccacctgag
     1801 aacgtgcgga tgaacagcgt gaacttcaag aacatcctcc agtgggagag ccccgccttc
     1861 gccaagggaa atctgacctt cacagcccag tacctgagct accggatctt ccaggacaag
     1921 tgcatgaaca ccacactgac cgagtgcgac ttcagcagcc tgtctaagta cggcgatcac
     1981 accctgagag tgcgggccga gtttgccgat gagcacagcg attgggtcaa catcacattc
     2041 tgccccgtgg acgacaccat catcggccca cctggaatgc aggttgaggt gctggccgat
     2101 agcctgcaca tgagatttct ggcccctaag atcgagaacg agtacgagac atggaccatg
     2161 aagaacgtct acaacagctg gacctacaac gtgcagtact ggaagaacgg caccgacgag
     2221 aagttccaga tcacccctca gtacgacttc gaggtgctgc ggaacctgga accttggacc
     2281 acctactgtg tgcaagtgcg gggcttcctg cctgacagaa acaaagctgg cgagtggagc
     2341 gagcctgtgt gcgagcagac aacccacgat gagacagtgc cctcttggat ggtggccgtg
     2401 atcctgatgg ccagcgtgtt catggtttgc ctggctctgc tgggctgctt cgctctgctt
     2461 tggtgcgtct acaagaaggg cgccgagagc ttgtttaagg ggccgcgtga ttacaacccg
     2521 atatcgagca ccatttgtca tttgacgaat gaatctgatg ggcacacaac atcgttgtat
     2581 ggtattggat ttggtccctt catcattaca aacaagcact tgtttagaag aaataatgga
     2641 acactgttgg tccaatcact acatggtgta ttcaaggtca agaacaccac gactttgcaa
     2701 caaagcctca ttgatgggag ggacatgata attattcgca tgcctaagga tttcccacca
     2761 tttcctcaaa agctgaaatt tagagagcca caaagggaag agcgcatatg tcttgtgaca
     2821 accaacttcc aaacttaggc ggccgcgttt aaacccgctg atcagcctcg actgtgcctt
     2881 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     2941 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     3001 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagtca
     3061 atagcaggca tgctggggat gcggtgggct ctatgggacg tcggaattgc aagttgggcc
     3121 gccctctggt aaggttggga agccctgcaa agtaaactgg atggctttct tgccgccaag
     3181 gatctgatgg cgcaggggat caagatctga tcaagagaca ggatgaggat cgtttcgcat
     3241 gattgaacaa gatggattgc acgcaggttc tccggcggct tgggtggaga ggctattcgg
     3301 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     3361 gcagggtcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     3421 agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cggctgtgct
     3481 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     3541 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     3601 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     3661 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     3721 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgta tgcccgacgg
     3781 cgaggatctc gtcgtgaccc acggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     3841 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     3901 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     3961 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     4021 cgagttcttc tgagcgggac tctggggttc gagaattggc ttggactcct gttgatagat
     4081 ccagtaatga cctcagaact ccatctggat ttgttcagaa cgctcggttg ccgccgggcg
     4141 ttttttattg gtgagaatcc aagcactagc catgaccaga cattgattat tgactagtta
     4201 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
     4261 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     4321 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
     4381 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     4441 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
     4501 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     4561 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
     4621 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
     4681 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
     4741 ggaggtctat ataagcagag ctctctggct aactagagaa cccactgctt actggcttat
     4801 cgaaattaat acgactcact atagggagtc ccaagctgac tagcgccacc atggtttcca
     4861 agggcgaaga ggacaatatg gccagcctgc ctgccacaca cgagctgcac atctttggca
     4921 gcatcaacgg cgtggacttc gacatggtcg gacaaggcac cggcaatccc aacgacggct
     4981 acgaggaact gaacctgaag tccacaaagg gcgacctcca gttcagcccc tggattctgg
     5041 tgcctcacat cggctacggc ttccaccagt acctgcctta tcctgacggc atgagccctt
     5101 tccaggccgc catggttgat ggctctggct accaggtcca ccggaccatg cagtttgagg
     5161 atggcgcttc cctgaccgtg aactacagat acacctacga gggcagccac atcaaaggcg
     5221 aggcccaagt gaaaggcaca ggcttccctg ctgatggccc cgtgatgacc aattctctga
     5281 cagccgccga ctggtgcaga agcaagaaaa cataccccaa cgacaagacc atcatcagca
     5341 ccttcaagtg gtcctacacc accggcaacg gcaagcggta cagaagcacc gccagaacca
     5401 cctacacctt cgccaaacct atggccgcca actacctgaa gaaccagcct atgtacgtgt
     5461 tccgcaagac cgagctgaag cactccaaga cagaactgaa cttcaaagag tggcagaaag
     5521 ccttcaccga cgtgatgggc atggacgaac tctacaaatg agcggccaag tttaaacccg
     5581 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     5641 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     5701 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     5761 caagggggag gattgggaag tcaatagcag gcatgctggg gatgcggtgg gctctatggc
     5821 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     5881 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     5941 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     6001 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     6061 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     6121 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     6181 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     6241 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     6301 tggaatgtgt gtcagttagg ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     6361 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     6421 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
     6481 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
     6541 tcggaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     6601 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     6661 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     6721 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     6781 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     6841 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     6901 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     6961 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     7021 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     7081 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     7141 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     7201 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     7261 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     7321 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     7381 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     7441 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     7501 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     7561 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     7621 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     7681 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     7741 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aa
//
