LOCUS       sequence-392621-        8538 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human TOLL protein expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8538)
  TITLE     TREAT-AD Plasmids 2023
REFERENCE   2  (bases 1 to 8538)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pDEST26-hTLR4-Nluc
FEATURES             Location/Qualifiers
     source          1..8538
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    677..701
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3228..3252)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3263..3289
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3320..3835
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(3875..3894)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3919..3967
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4169..4597
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4256..4275)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4466..4487
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4606..4626)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4611..4940
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4791..4926
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4853..4872
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5007..5801
                     /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(5061..5080)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5671..5690
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5977..6098
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6014..6033)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6068..6087
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6147..6163)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6147..6163)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6160..6182)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6171..6187
                     /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(6195..6201,6202..6219,6220..6225))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6240..6261
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6378..6395)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6549..7137)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6629..6648)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7308..8099,8100..8168))
                     /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     7931..7950
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8169..8273)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8348..8367)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 gctagttaag ctagcaacaa gtttgtacaa aaaagcaggc tccaccatga tgtctgcctc
      721 gcgcctggct gggactctga tcccagccat ggccttcctc tcctgcgtga gaccagaaag
      781 ctgggagccc tgcgtggagg tggttcctaa tattacttat caatgcatgg agctgaattt
      841 ctacaaaatc cccgacaacc tccccttctc aaccaagaac ctggacctga gctttaatcc
      901 cctgaggcat ttaggcagct atagcttctt cagtttccca gaactgcagg tgctggattt
      961 atccaggtgt gaaatccaga caattgaaga tggggcatat cagagcctaa gccacctctc
     1021 taccttaata ttgacaggaa accccatcca gagtttagcc ctgggagcct tttctggact
     1081 atcaagttta cagaagctgg tggctgtgga gacaaatcta gcatctctag agaacttccc
     1141 cattggacat ctcaaaactt tgaaagaact taatgtggct cacaatctta tccaatcttt
     1201 caaattacct gagtattttt ctaatctgac caatctagag cacttggacc tttccagcaa
     1261 caagattcaa agtatttatt gcacagactt gcgggttcta catcaaatgc ccctactcaa
     1321 tctctcttta gacctgtccc tgaaccctat gaactttatc caaccaggtg catttaaaga
     1381 aattaggctt cataagctga ctttaagaaa taattttgat agtttaaatg taatgaaaac
     1441 ttgtattcaa ggtctggctg gtttagaagt ccatcgtttg gttctgggag aatttagaaa
     1501 tgaaggaaac ttggaaaagt ttgacaaatc tgctctagag ggcctgtgca atttgaccat
     1561 tgaagaattc cgattagcat acttagacta ctacctcgat gatattattg acttatttaa
     1621 ttgtttgaca aatgtttctt cattttccct ggtgagtgtg actattgaaa gggtaaaaga
     1681 cttttcttat aatttcggat ggcaacattt agaattagtt aactgtaaat ttggacagtt
     1741 tcccacattg aaactcaaat ctctcaaaag gcttactttc acttccaaca aaggtgggaa
     1801 tgctttttca gaagttgatc taccaagcct tgagtttcta gatctcagta gaaatggctt
     1861 gagtttcaaa ggttgctgtt ctcaaagtga ttttgggaca accagcctaa agtatttaga
     1921 tctgagcttc aatggtgtta ttaccatgag ttcaaacttc ttgggcttag aacaactaga
     1981 acatctggat ttccagcatt ccaatttgaa acaaatgagt gagttttcag tattcctatc
     2041 actcagaaac ctcatttacc ttgacatttc tcatactcac accagagttg ctttcaatgg
     2101 catcttcaat ggcttgtcca gtctcgaagt cttgaaaatg gctggcaatt ctttccagga
     2161 aaacttcctt ccagatatct tcacagagct gagaaacttg accttcctgg acctctctca
     2221 gtgtcaactg gagcagttgt ctccaacagc atttaactca ctctccagtc ttcaggtact
     2281 aaatatgagc cacaacaact tcttttcatt ggatacgttt ccttataagt gtctgaactc
     2341 cctccaggtt cttgattaca gtctcaatca cataatgact tccaaaaaac aggaactaca
     2401 gcattttcca agtagtctag ctttcttaaa tcttactcag aatgactttg cttgtacttg
     2461 tgaacaccag agtttcctgc aatggatcaa ggaccagagg cagctcttgg tggaagttga
     2521 acgaatggaa tgtgcaacac cttcagataa gcagggcatg cctgtgctga gtttgaatat
     2581 cacctgtcag atgaataaga ccatcattgg tgtgtcggtc ctcagtgtgc ttgtagtatc
     2641 tgttgtagca gttctggtct ataagttcta ttttcacctg atgcttcttg ctggctgcat
     2701 aaagtatggt agaggtgaaa acatctatga tgcctttgtt atctactcaa gccaggatga
     2761 ggactgggta aggaatgagc tagtaaagaa tttagaagaa ggggtgcctc catttcagct
     2821 ctgccttcac tacagagact ttattcccgg tgtggccatt gctgccaaca tcatccatga
     2881 aggtttccat aaaagccgaa aggtgattgt tgtggtgtcc cagcacttca tccagagccg
     2941 ctggtgtatc tttgaatatg agattgctca gacctggcag tttctgagca gtcgtgctgg
     3001 tatcatcttc attgtcctgc agaaggtgga gaagaccctg ctcaggcagc aggtggagct
     3061 gtaccgcctt ctcagcagga acacttacct ggagtgggag gacagtgtcc tggggcggca
     3121 catcttctgg agacgactca gaaaagccct gctggatggt aaatcatgga atccagaagg
     3181 aacagtgggt acaggatgca attggcagga agcaacatct atcttggacc cagctttctt
     3241 gtacaaagtg gttgatctcg agtacccata cgatgttcca gattacgctg gagggggtgg
     3301 atctggaggg ggtggatcta tggtcttcac actcgaagat ttcgttgggg actggcgaca
     3361 gacagccggc tacaacctgg accaagtcct tgaacaggga ggtgtgtcca gtttgtttca
     3421 gaatctcggg gtgtccgtaa ctccgatcca aaggattgtc ctgagcggtg aaaatgggct
     3481 gaagatcgac atccatgtca tcatcccgta tgaaggtctg agcggcgacc aaatgggcca
     3541 gatcgaaaaa atttttaagg tggtgtaccc tgtggatgat catcacttta aggtgatcct
     3601 gcactatggc acactggtaa tcgacggggt tacgccgaac atgatcgact atttcggacg
     3661 gccgtatgaa ggcatcgccg tgttcgacgg caaaaagatc actgtaacag ggaccctgtg
     3721 gaacggcaac aaaattatcg acgagcgcct gatcaacccc gacggctccc tgctgttccg
     3781 agtaaccatc aacggagtga ccggctggcg gctgtgcgaa cgcattctgg cgtgaggtac
     3841 cggttagtaa tgagtttaaa cgggggaggc taactgaaac acggaaggag acaataccgg
     3901 aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggg tgttgggtcg
     3961 tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga
     4021 ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg
     4081 ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat agcagatctg
     4141 cgcagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     4201 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     4261 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4321 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4381 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     4441 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     4501 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     4561 aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt
     4621 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     4681 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     4741 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     4801 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4861 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     4921 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc
     4981 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     5041 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     5101 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     5161 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     5221 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     5281 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     5341 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     5401 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     5461 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     5521 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     5581 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     5641 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     5701 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     5761 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcgc
     5821 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     5881 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     5941 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     6001 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     6061 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     6121 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     6181 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     6241 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     6301 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     6361 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     6421 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     6481 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     6541 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     6601 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     6661 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     6721 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     6781 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     6841 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     6901 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     6961 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     7021 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     7081 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     7141 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     7201 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     7261 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     7321 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     7381 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     7441 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     7501 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     7561 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     7621 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     7681 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     7741 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     7801 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     7861 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     7921 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     7981 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     8041 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     8101 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     8161 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     8221 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     8281 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca
     8341 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
     8401 tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
     8461 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
     8521 gggccagata tacgcgtt
//
