LOCUS       sequence-487917-        8389 bp    DNA     circular SYN 02-OCT-2025
DEFINITION  Constitutive expression of a MESA CTEVp chain encoding (N to C): 
            IL10RaSS-3xFLAG-IL10RaECD-IL10RaTMD-CTEVp(190K)-PRS(M)-VP64-ZF6a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8389)
  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 8389)
  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: L4315 IL-10Ra CTEVp chain in PolyTX-mTagBFP2
FEATURES             Location/Qualifiers
     source          1..8389
                     /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"
     CDS             1717..1782
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2887..2907
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQM"
     CDS             2926..2946
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2953..3102
                     /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    3466..3690
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             3833..4627
                     /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(3887..3906)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4497..4516
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      4658..4752
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     enhancer        4773..5152
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5153..5356
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5307..5327
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5401..5420
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5401..5419
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      5439..5448
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    6192..6416
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6462..6890
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6549..6568)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6759..6780
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6913..7109
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6960..7095
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7022..7041
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    7229..7350
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7266..7285)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7320..7339
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7399..7415)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7399..7415)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7412..7434)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7423..7439
                     /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(7447..7453,7454..7471,7472..7477))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7492..7513
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7630..7647)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7801..8389)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7881..7900)
                     /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 accatgctgc cctgcctggt tgttctgctt
     1681 gccgctctgc tgtctctgag actgggatct gacgccgact acaaggacca cgacggcgat
     1741 tataaggatc atgacatcga ttacaaggac gatgacgaca agcacggcac cgagctgcct
     1801 tctccaccta gcgtttggtt cgaggccgag ttcttccacc acatcctgca ctggacccct
     1861 attcctaacc agagcgagag cacctgttac gaggtggccc tgctgagata cggcatcgag
     1921 agctggaaca gcatcagcaa ctgcagccag acactgagct acgacctgac cgccgtgaca
     1981 ctggatctgt accacagcaa cggctaccgg gccagagtta gagccgtgga tggaagcaga
     2041 cactccaact ggaccgtgac caacaccaga ttcagcgtgg acgaagtgac cctgacagtg
     2101 ggcagcgtga acctggaaat ccacaacggc ttcatcctgg gcaagatcca gctgcctcgg
     2161 cctaagatgg cccctgccaa tgatacctac gagagcatct tcagccactt ccgcgagtac
     2221 gagatcgcca tcagaaaggt gcccggcaac ttcaccttca cacacaagaa agtgaagcac
     2281 gagaacttca gcctgctgac ctctggcgaa gtgggcgagt tctgcgtgca agtgaaaccc
     2341 tctgtggcca gccggtccaa caaaggcatg tggtccaaag aggaatgcat cagcctgacc
     2401 agacagtact tcaccgtgac aaacgtgatc atcttcttcg ccttcgtgct gctgctgtct
     2461 ggcgctctgg cttattgtct ggccctgcag ctgtacgtgc ggcggagaaa gaagggcgcc
     2521 aagagcatgt ctagcatggt gtcagacact agttgcacat tcccttcatc tgatggcata
     2581 ttctggaagc attggattca aaccaaggat gggcagtgtg gcagtccatt agtatcaact
     2641 agagatgggt tcattgttgg tatacactca gcatcgaatt tcaccaacac aaacaattat
     2701 ttcacaagcg tgccgaaaaa cttcatggaa ttgaagacaa atcaggaggc gcagcagtgg
     2761 gttagtggtt ggcgattaaa tgctgactca gtattgtggg ggggccataa agttttcatg
     2821 gtgaaacctg aagagccttt tcagccagtt aaggaagcga ctcaactcat gaatcttaag
     2881 ggctcggaga acctgtattt tcagatgggg ggaggcagcg ggggccctaa gaaaaagcgc
     2941 aaagtctccg gagacgctct ggatgacttt gatctggaca tgcttggttc cgatgccttg
     3001 gacgattttg acctcgatat gctgggctcc gacgctctcg acgatttcga cctcgacatg
     3061 ctgggtagcg atgcactgga tgactttgat ctggatatgc tcggatccgg tggtggcggg
     3121 tctggaggag gaggtagtgg tggaggtggc tctggtaccg ctagacccgg cgaaagacct
     3181 ttccagtgcc ggatctgcat gaggaacttc agcaagggcg agagactcgt gcggcacacc
     3241 agaacacaca caggcgagaa gcccttccag tgtagaatct gtatgcgcaa cttcagccgg
     3301 atggacaacc tgagcaccca cctgagaacc cataccgggg agaagccatt tcaatgccgc
     3361 atctgtatga gaaatttttc ccggaaggac gccctgaacc ggcacctgaa aacacacctg
     3421 agaggcagct gagcggccgc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     3481 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     3541 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     3601 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gtcaatagca
     3661 ggcatgctgg ggatgcggtg ggctctatgg gacgtcggaa ttgcaagttg ggccgccctc
     3721 tggtaaggtt gggaagccct gcaaagtaaa ctggatggct ttcttgccgc caaggatctg
     3781 atggcgcagg ggatcaagat ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     3841 acaagatgga ttgcacgcag gttctccggc ggcttgggtg gagaggctat tcggctatga
     3901 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     3961 tcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga
     4021 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcggctg tgctcgacgt
     4081 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     4141 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     4201 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     4261 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     4321 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgtatgcccg acggcgagga
     4381 tctcgtcgtg acccacggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     4441 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     4501 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     4561 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     4621 cttctgagcg ggactctggg gttcgagaat tggcttggac tcctgttgat agatccagta
     4681 atgacctcag aactccatct ggatttgttc agaacgctcg gttgccgccg ggcgtttttt
     4741 attggtgaga atccaagcac tagccatgac cagacattga ttattgacta gttattaata
     4801 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
     4861 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
     4921 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     4981 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
     5041 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
     5101 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
     5161 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     5221 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     5281 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
     5341 ctatataagc agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat
     5401 taatacgact cactataggg agtcccaagc tgactagcgc caccatggtt tccaagggcg
     5461 aagaactgat caaagaaaac atgcacatga agctctacat ggaaggcacc gtggacaacc
     5521 accacttcaa gtgtaccagc gaaggcgagg gcaagcctta cgagggcacc cagaccatga
     5581 gaatcaaggt ggtggaaggc ggccctctgc ctttcgcctt tgatatcctg gccaccagct
     5641 ttctgtacgg cagcaagacc ttcatcaatc acacccaggg catccccgat ttcttcaagc
     5701 agagcttccc cgagggcttc acctgggaga gagtgaccac atacgaggat ggcggcgtgc
     5761 tgacagccac acaggataca agcctccagg acggctgcct gatctacaac gtgaagatcc
     5821 ggggcgtgaa cttcaccagc aacggccccg tgatgcagaa gaaaaccctt ggctgggaag
     5881 ccttcaccga gacactgtat cctgccgatg gcggcctgga aggcagaaac gatatggccc
     5941 tgaagctcgt cggcggctct cacctgattg ccaatgccaa gaccacctac agaagcaaga
     6001 agcccgccaa gaacctgaag atgcccggcg tgtactacgt ggactaccgg ctggaaagaa
     6061 tcaaagaggc caacaacgag acatacgtgg aacagcacga ggtggccgtg gccagatact
     6121 gtgatctgcc ttctaagctg ggccacaagc tgaactgagc ggccaagttt aaacccgctg
     6181 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     6241 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     6301 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     6361 gggggaggat tgggaagtca atagcaggca tgctggggat gcggtgggct ctatggcttc
     6421 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     6481 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     6541 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     6601 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     6661 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     6721 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     6781 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     6841 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     6901 aatgtgtgtc agttaggctc aattagtcag caaccatagt cccgccccta actccgccca
     6961 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     7021 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag
     7081 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg
     7141 gaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     7201 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     7261 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     7321 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     7381 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     7441 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     7501 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     7561 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     7621 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     7681 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     7741 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     7801 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     7861 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     7921 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     7981 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     8041 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     8101 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     8161 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     8221 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     8281 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     8341 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaa
//
