LOCUS       sequence-487920-        7198 bp    DNA     circular SYN 02-OCT-2025
DEFINITION  Constitutive expression of VP64-intN split synTF component.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7198)
  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 7198)
  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: L4872 VP64-intN in PolyTX-mNeonGreen
FEATURES             Location/Qualifiers
     source          1..7198
                     /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      1642..1651
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1651..1716
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1723..1743
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1759..1908
                     /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    2278..2502
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             2645..3439
                     /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(2699..2718)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3309..3328
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      3470..3564
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     enhancer        3585..3964
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3965..4168
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4119..4139
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4213..4232
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4213..4231
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4251..4260
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      4512..4521
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    5001..5225
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5271..5699
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5358..5377)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5568..5589
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5722..5918
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5769..5904
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5831..5850
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    6038..6159
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6075..6094)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6129..6148
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6208..6224)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6208..6224)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6221..6243)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6232..6248
                     /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(6256..6262,6263..6280,6281..6286))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6301..6322
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6439..6456)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6610..7198)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6690..6709)
                     /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 cgccaccatg gactataaag accatgacgg tgactacaag
     1681 gatcacgaca tagattataa agacgatgac gacaagatgg cacctaagaa gaagcgcaaa
     1741 gttggctctg gcgaattcga cgcccttgat gactttgact tggacatgct ggggtcagac
     1801 gcactcgatg attttgacct tgatatgctg ggaagcgatg cacttgatga ctttgacttg
     1861 gacatgctcg gctccgacgc tcttgatgat ttcgatcttg acatgctcgg atccggtggt
     1921 ggcgggtctg gaggaggagg tactggtgga ggtggcactg gtaccagaag cggatactgc
     1981 ctggacctga aaacccaggt gcagacccct cagggcatga aggagatcag caacatccag
     2041 gtgggcgacc tggtgctgag caacaccggc tacaacgagg tgctgaacgt gttccccaag
     2101 agcaagaaga agagctacaa gatcaccctg gaggacggca aggagatcat ctgcagcgag
     2161 gagcacctgt tccccaccca gaccggcgag atgaacatca gcggcggcct gaaggagggc
     2221 atgtgcctgt acgtgaagga gtgagcggcc gcgtttaaac ccgctgatca gcctcgactg
     2281 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2341 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     2401 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     2461 aagtcaatag caggcatgct ggggatgcgg tgggctctat gggacgtcgg aattgcaagt
     2521 tgggccgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc
     2581 gccaaggatc tgatggcgca ggggatcaag atctgatcaa gagacaggat gaggatcgtt
     2641 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gcggcttggg tggagaggct
     2701 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     2761 gtcagcgcag ggtcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     2821 actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcggc
     2881 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     2941 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     3001 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     3061 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     3121 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgtatgcc
     3181 cgacggcgag gatctcgtcg tgacccacgg cgatgcctgc ttgccgaata tcatggtgga
     3241 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     3301 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     3361 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     3421 tcttgacgag ttcttctgag cgggactctg gggttcgaga attggcttgg actcctgttg
     3481 atagatccag taatgacctc agaactccat ctggatttgt tcagaacgct cggttgccgc
     3541 cgggcgtttt ttattggtga gaatccaagc actagccatg accagacatt gattattgac
     3601 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
     3661 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     3721 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
     3781 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
     3841 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
     3901 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
     3961 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
     4021 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
     4081 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
     4141 acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg
     4201 gcttatcgaa attaatacga ctcactatag ggagtcccaa gctgactagc gccaccatgg
     4261 tttccaaggg cgaagaggac aatatggcca gcctgcctgc cacacacgag ctgcacatct
     4321 ttggcagcat caacggcgtg gacttcgaca tggtcggaca aggcaccggc aatcccaacg
     4381 acggctacga ggaactgaac ctgaagtcca caaagggcga cctccagttc agcccctgga
     4441 ttctggtgcc tcacatcggc tacggcttcc accagtacct gccttatcct gacggcatga
     4501 gccctttcca ggccgccatg gttgatggct ctggctacca ggtccaccgg accatgcagt
     4561 ttgaggatgg cgcttccctg accgtgaact acagatacac ctacgagggc agccacatca
     4621 aaggcgaggc ccaagtgaaa ggcacaggct tccctgctga tggccccgtg atgaccaatt
     4681 ctctgacagc cgccgactgg tgcagaagca agaaaacata ccccaacgac aagaccatca
     4741 tcagcacctt caagtggtcc tacaccaccg gcaacggcaa gcggtacaga agcaccgcca
     4801 gaaccaccta caccttcgcc aaacctatgg ccgccaacta cctgaagaac cagcctatgt
     4861 acgtgttccg caagaccgag ctgaagcact ccaagacaga actgaacttc aaagagtggc
     4921 agaaagcctt caccgacgtg atgggcatgg acgaactcta caaatgagcg gccaagttta
     4981 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     5041 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     5101 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     5161 ggacagcaag ggggaggatt gggaagtcaa tagcaggcat gctggggatg cggtgggctc
     5221 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     5281 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     5341 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     5401 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     5461 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     5521 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     5581 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     5641 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
     5701 attctgtgga atgtgtgtca gttaggctca attagtcagc aaccatagtc ccgcccctaa
     5761 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     5821 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     5881 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     5941 ccattttcgg aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     6001 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     6061 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     6121 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     6181 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     6241 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     6301 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     6361 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     6421 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     6481 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     6541 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     6601 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     6661 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     6721 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     6781 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     6841 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     6901 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     6961 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     7021 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     7081 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     7141 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaa
//
