LOCUS       sequence-489480-        8662 bp    DNA     circular SYN 16-OCT-2025
DEFINITION  Expression of a MESA CTEVp chain encoding (N to C): 
            IL10RaSS-3xFLAG-IL10RaECD-IL10RaTMD-CTEVp(190K)-PRS(M)-hTbet.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8662)
  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 8662)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 16, 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: L4428 IL-10Ra CTEVp chain with Tbet in TUPV3
FEATURES             Location/Qualifiers
     source          1..8662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        350..1532
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          581..1523
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1480..1500
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1534..1558
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1708..1773
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2878..2898
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQM"
     primer_bind     complement(4569..4586)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4575..4799
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5568..5996
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5655..5674)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5865..5886
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6019..6215
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6066..6201
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6128..6147
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    6335..6456
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6372..6391)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6426..6445
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6505..6521)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6505..6521)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6518..6540)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6529..6545
                     /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(6553..6559,6560..6577,6578..6583))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6598..6619
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6736..6753)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6907..7495)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6987..7006)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7666..8457,8458..8526))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     8289..8308
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8527..8631)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatcgaag acggactaaa tagtagttca gacgccgtta agcgcaagat
       61 ctggggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
      121 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
      181 gggccagata tacgcgtgag taattcatac aaaaggactc gcccctgcct tggggaatcc
      241 cagggaccgt cgttaaactc ccactaacgt agaacccaga gatcgctgcg ttcccgcccc
      301 ctcacccgcc cgctctcgtc atcactgagg tggagaagag catgcgtgag gctccggtgc
      361 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
      421 caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
      481 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
      541 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc
      601 ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccacgcc
      661 cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt
      721 cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcttgggc
      781 gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata
      841 agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata
      901 gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg
      961 gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc
     1021 caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg
     1081 cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt
     1141 gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc
     1201 gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag
     1261 ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct
     1321 cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc
     1381 cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct
     1441 tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc
     1501 aaagtttttt tcttccattt caggtgtcgt gagagctcgt ttagtgaacc gtcagatcgc
     1561 ctggagacgc catccacgct gttttgacct ccatagaaga gaacgggacc gatccaaatt
     1621 ctcgaggcta gctgtacagc caccatgctg ccctgcctgg ttgttctgct tgccgctctg
     1681 ctgtctctga gactgggatc tgacgccgac tacaaggacc acgacggcga ttataaggat
     1741 catgacatcg attacaagga cgatgacgac aagcacggca ccgagctgcc ttctccacct
     1801 agcgtttggt tcgaggccga gttcttccac cacatcctgc actggacccc tattcctaac
     1861 cagagcgaga gcacctgtta cgaggtggcc ctgctgagat acggcatcga gagctggaac
     1921 agcatcagca actgcagcca gacactgagc tacgacctga ccgccgtgac actggatctg
     1981 taccacagca acggctaccg ggccagagtt agagccgtgg atggaagcag acactccaac
     2041 tggaccgtga ccaacaccag attcagcgtg gacgaagtga ccctgacagt gggcagcgtg
     2101 aacctggaaa tccacaacgg cttcatcctg ggcaagatcc agctgcctcg gcctaagatg
     2161 gcccctgcca atgataccta cgagagcatc ttcagccact tccgcgagta cgagatcgcc
     2221 atcagaaagg tgcccggcaa cttcaccttc acacacaaga aagtgaagca cgagaacttc
     2281 agcctgctga cctctggcga agtgggcgag ttctgcgtgc aagtgaaacc ctctgtggcc
     2341 agccggtcca acaaaggcat gtggtccaaa gaggaatgca tcagcctgac cagacagtac
     2401 ttcaccgtga caaacgtgat catcttcttc gccttcgtgc tgctgctgtc tggcgctctg
     2461 gcttattgtc tggccctgca gctgtacgtg cggcggagaa agaagggcgc caagagcatg
     2521 tctagcatgg tgtcagacac tagttgcaca ttcccttcat ctgatggcat attctggaag
     2581 cattggattc aaaccaagga tgggcagtgt ggcagtccat tagtatcaac tagagatggg
     2641 ttcattgttg gtatacactc agcatcgaat ttcaccaaca caaacaatta tttcacaagc
     2701 gtgccgaaaa acttcatgga attgaaaaca aatcaggagg cgcagcagtg ggttagtggt
     2761 tggcgattaa atgctgactc agtattgtgg gggggccata aagttttcat ggtgaaacct
     2821 gaagagcctt ttcagccagt taaggaagcg actcaactca tgaatcttaa gggctcggag
     2881 aacctgtatt ttcagatggg gggaggcagc gggggcatgg gcatcgtgga acctggctgt
     2941 ggcgatatgc tgaccggcac agaacctatg cctggctccg atgaaggcag agcacctggt
     3001 gctgatcccc agcacagata cttctaccct gagcctggcg ctcaggacgc cgatgagaga
     3061 agaggcggag gatctctggg cagcccttat cctggcggag cacttgttcc agctcctcca
     3121 agcagattcc tgggagccta cgcctatcct cctagacctc aggccgctgg atttcctggc
     3181 gctggcgaat catttcctcc accagccgat gccgagggct atcaacctgg cgaaggatac
     3241 gctgcccctg atcctagagc cggcctttat cctggaccta gagaggacta cgccctgcct
     3301 gctggactgg aagtgtctgg caaactgaga gtggccctga acaaccatct gctgtggtcc
     3361 aagttcaacc agcaccagac cgagatgatc attaccaagc agggccgcag aatgttcccc
     3421 ttcctgtcct ttaccgtggc cggcctggaa cctaccagcc actacaggat gttcgtggac
     3481 gtggtcctgg tggaccagca tcattggcgg taccagagcg gcaaatgggt gcagtgtgga
     3541 aaggccgaag gcagcatgcc cggcaacaga ctgtatgtgc accctgacag ccccaacaca
     3601 ggcgctcact ggatgagaca agaagtgtcc ttcggcaagc tgaagctgac caacaacaag
     3661 ggcgcttcca acaacgtgac ccagatgatc gtgctgcaga gcctgcacaa gtaccagcct
     3721 agactgcaca ttgtggaagt gaacgacggc gagcctgagg ccgcctgtaa tgcctctaac
     3781 acccacatct tcaccttcca agagacacag tttatcgccg tgaccgccta ccagaacgcc
     3841 gagatcacac agctgaagat cgacaacaac cccttcgcca agggcttccg cgagaacttc
     3901 gagagcatgt ataccagcgt ggacaccagc attccctctc cacctggacc aaactgccag
     3961 ttcctcggcg gcgatcacta tagccctctg ctgcccaatc agtaccccgt gcctagcaga
     4021 ttctaccccg atctgcctgg acaggccaag gatgtggtgc ctcaggcata ttggctggga
     4081 gcccctagag atcacagcta cgaggccgag tttcgggccg tgtctatgaa gcctgccttc
     4141 ctgccttctg ctcccggacc taccatgagc tactacagag gccaagaggt gctggctcca
     4201 ggtgctggat ggcctgttgc tcctcagtac cctcctaaga tgggacccgc cagctggttc
     4261 agacccatga gaaccctgcc tatggaacca ggacctggcg gatctgaagg caggggacct
     4321 gaagatcagg gacctccact cgtgtggaca gagatcgccc ctatcagacc cgagagcagc
     4381 gattctggac tcggcgaggg cgatagcaag cggagaagag tgtcccctta tccaagcagc
     4441 ggcgacagca gttctcctgc tggtgcccca tctccattcg acaaagaagc cgagggccag
     4501 ttctacaact acttccccaa ctgagcggcc gctcgagtct agagggcccg tttaaacccg
     4561 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     4621 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     4681 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     4741 caagggggag gattgggaag tcaatagcag gcatgctggg gatgcggtgg gctctatggc
     4801 ttctgaggca ggtcgaagag actgcccgac acatcttagt gagcgcatcg cgataagctc
     4861 tagagggaca gccccccccc aaagccccca gggatgtaat tacgtccctc ccccgctagg
     4921 gggcagcagc gagccgcccg gggctccgct ccggtccggc gctccccccg catccccgag
     4981 ccggcagcgt gcggggacag cccgggcacg gggaaggtgg cacgggatcg ctttcctctg
     5041 aacgcttctc gctgctcttt gagcctgcag acacctgggg ggatacgggg aaaaagcttt
     5101 aggctgaaag agagatttag aatgacagaa tcatagaacg gcctgcgtac ggatggggct
     5161 ctagctctag agcttgatat cgaattcctg cagccccgct agagggacag ccccccccca
     5221 aagcccccag ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg
     5281 ggctccgctc cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc
     5341 ccgggcacgg ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg
     5401 agcctgcaga cacctggggg gatacgggga aaaagcttta ggctgaaaga gagatttaga
     5461 atgacagaat catagaacgg cctgggcgcg cctggtggcg aacgatcaac agacgggaga
     5521 ggttacaagt cttcgctgac agcgaccgga ctctaggggg tatccccacg cgccctgtag
     5581 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     5641 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     5701 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     5761 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     5821 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     5881 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     5941 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     6001 ctgtggaatg tgtgtcagtt aggctcaatt agtcagcaac catagtcccg cccctaactc
     6061 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     6121 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     6181 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     6241 ttttcggagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     6301 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     6361 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     6421 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     6481 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     6541 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     6601 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     6661 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     6721 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     6781 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     6841 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     6901 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     6961 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     7021 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     7081 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     7141 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     7201 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     7261 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     7321 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     7381 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     7441 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     7501 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     7561 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     7621 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     7681 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     7741 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     7801 accgcgagat ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     7861 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     7921 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     7981 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     8041 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     8101 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     8161 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     8221 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     8281 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     8341 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     8401 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     8461 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     8521 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     8581 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     8641 ccgaaaagtg ccacctgacg tc
//
