LOCUS       sequence-487901-        7786 bp    DNA     circular SYN 02-OCT-2025
DEFINITION  Constitutive expression of a MESA NTEVp chain encoding (N to C): 
            hIgGVHSS-3xFLAG-IL10RbECD-IL10RbTMD-NTEVp(75S).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7786)
  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 7786)
  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: L4308 IL-10Rb NTEVp chain (human IgG VH SS) in...
FEATURES             Location/Qualifiers
     source          1..7786
                     /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             1711..1776
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    2866..3090
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             3233..4027
                     /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(3287..3306)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3897..3916
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      4058..4152
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     enhancer        4173..4552
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4553..4756
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4707..4727
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4801..4820
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4801..4819
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4839..4848
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      5100..5109
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    5589..5813
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5859..6287
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5946..5965)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6156..6177
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6310..6506
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6357..6492
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6419..6438
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    6626..6747
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6663..6682)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6717..6736
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6796..6812)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6796..6812)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6809..6831)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6820..6836
                     /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(6844..6850,6851..6868,6869..6874))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6889..6910
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7027..7044)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7198..7786)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7278..7297)
                     /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 accatggagt ttggcctgag ctggctgttt
     1681 ctggtggcca ttctgaaggg cgtgcagtgc gactacaagg accacgacgg cgattataag
     1741 gatcatgaca tcgattacaa ggacgatgac gacaagatgg tgcctccacc tgagaacgtg
     1801 cggatgaaca gcgtgaactt caagaacatc ctccagtggg agagccccgc cttcgccaag
     1861 ggaaatctga ccttcacagc ccagtacctg agctaccgga tcttccagga caagtgcatg
     1921 aacaccacac tgaccgagtg cgacttcagc agcctgtcta agtacggcga tcacaccctg
     1981 agagtgcggg ccgagtttgc cgatgagcac agcgattggg tcaacatcac attctgcccc
     2041 gtggacgaca ccatcatcgg cccacctgga atgcaggttg aggtgctggc cgatagcctg
     2101 cacatgagat ttctggcccc taagatcgag aacgagtacg agacatggac catgaagaac
     2161 gtctacaaca gctggaccta caacgtgcag tactggaaga acggcaccga cgagaagttc
     2221 cagatcaccc ctcagtacga cttcgaggtg ctgcggaacc tggaaccttg gaccacctac
     2281 tgtgtgcaag tgcggggctt cctgcctgac agaaacaaag ctggcgagtg gagcgagcct
     2341 gtgtgcgagc agacaaccca cgatgagaca gtgccctctt ggatggtggc cgtgatcctg
     2401 atggccagcg tgttcatggt ttgcctggct ctgctgggct gcttcgctct gctttggtgc
     2461 gtctacaaga agggcgccga gagcttgttt aaggggccgc gtgattacaa cccgatatcg
     2521 agcaccattt gtcatttgac gaatgaatct gatgggcaca caacatcgtt gtatggtatt
     2581 ggatttggtc ccttcatcat tacaaacaag cacttgttta gaagaaataa tggaacactg
     2641 ttggtccaat cactacatgg tgtattcaag gtcaagaaca ccacgacttt gcaacaaagc
     2701 ctcattgatg ggagggacat gataattatt cgcatgccta aggatttccc accatttcct
     2761 caaaagctga aatttagaga gccacaaagg gaagagcgca tatgtcttgt gacaaccaac
     2821 ttccaaactt aggcggccgc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     2881 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     2941 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     3001 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gtcaatagca
     3061 ggcatgctgg ggatgcggtg ggctctatgg gacgtcggaa ttgcaagttg ggccgccctc
     3121 tggtaaggtt gggaagccct gcaaagtaaa ctggatggct ttcttgccgc caaggatctg
     3181 atggcgcagg ggatcaagat ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     3241 acaagatgga ttgcacgcag gttctccggc ggcttgggtg gagaggctat tcggctatga
     3301 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     3361 tcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga
     3421 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcggctg tgctcgacgt
     3481 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     3541 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     3601 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     3661 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     3721 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgtatgcccg acggcgagga
     3781 tctcgtcgtg acccacggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     3841 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     3901 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     3961 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     4021 cttctgagcg ggactctggg gttcgagaat tggcttggac tcctgttgat agatccagta
     4081 atgacctcag aactccatct ggatttgttc agaacgctcg gttgccgccg ggcgtttttt
     4141 attggtgaga atccaagcac tagccatgac cagacattga ttattgacta gttattaata
     4201 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
     4261 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
     4321 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     4381 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
     4441 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
     4501 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
     4561 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     4621 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     4681 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
     4741 ctatataagc agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat
     4801 taatacgact cactataggg agtcccaagc tgactagcgc caccatggtt tccaagggcg
     4861 aagaggacaa tatggccagc ctgcctgcca cacacgagct gcacatcttt ggcagcatca
     4921 acggcgtgga cttcgacatg gtcggacaag gcaccggcaa tcccaacgac ggctacgagg
     4981 aactgaacct gaagtccaca aagggcgacc tccagttcag cccctggatt ctggtgcctc
     5041 acatcggcta cggcttccac cagtacctgc cttatcctga cggcatgagc cctttccagg
     5101 ccgccatggt tgatggctct ggctaccagg tccaccggac catgcagttt gaggatggcg
     5161 cttccctgac cgtgaactac agatacacct acgagggcag ccacatcaaa ggcgaggccc
     5221 aagtgaaagg cacaggcttc cctgctgatg gccccgtgat gaccaattct ctgacagccg
     5281 ccgactggtg cagaagcaag aaaacatacc ccaacgacaa gaccatcatc agcaccttca
     5341 agtggtccta caccaccggc aacggcaagc ggtacagaag caccgccaga accacctaca
     5401 ccttcgccaa acctatggcc gccaactacc tgaagaacca gcctatgtac gtgttccgca
     5461 agaccgagct gaagcactcc aagacagaac tgaacttcaa agagtggcag aaagccttca
     5521 ccgacgtgat gggcatggac gaactctaca aatgagcggc caagtttaaa cccgctgatc
     5581 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     5641 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     5701 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     5761 ggaggattgg gaagtcaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     5821 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     5881 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     5941 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     6001 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     6061 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     6121 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     6181 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     6241 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     6301 gtgtgtcagt taggctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     6361 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     6421 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
     6481 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggag
     6541 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     6601 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     6661 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     6721 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     6781 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     6841 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     6901 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     6961 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     7021 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     7081 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     7141 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     7201 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     7261 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     7321 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     7381 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     7441 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     7501 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     7561 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     7621 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     7681 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     7741 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaa
//
