LOCUS       sequence-426572-        7971 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Produces the Chikungunya virus nonstructural protein 4 with a 
            c-terminal 3xFLAG tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7971)
  AUTHORS   Reyes Ballista JM, Hoover AJ, Noble JT, Acciani MD, Miazgowicz KL, 
            Harrison SA, Tabscott GAL, Duncan A, Barnes DN, Jimenez AR, Brindley
            MA
  TITLE     Chikungunya virus release is reduced by TIM-1 receptors through 
            binding of envelope phosphatidylserine.
  JOURNAL   J Virol. 2024 Jul 15:e0077524. doi: 10.1128/jvi.00775-24.
  PUBMED    39007616
REFERENCE   2  (bases 1 to 7971)
  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"; journalName: "J Virol. 
            2024 Jul 15:e0077524. doi: 10.1128/jvi.00775-24."
COMMENT     Sequence Label: pcDNAintron-CHIKV-nsp4-c3xFLAG
FEATURES             Location/Qualifiers
     source          1..7971
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     325..344
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        325..343
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      511..520
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2179..2244
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2321..2338)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2327..2551
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2597..3025
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2684..2703)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2894..2915
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3034..3054)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3039..3368
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3219..3354
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3281..3300
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3435..4229
                     /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(3489..3508)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4099..4118
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4403..4524
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4440..4459)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4494..4513
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4573..4589)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4573..4589)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4586..4608)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4597..4613
                     /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(4621..4627,4628..4645,4646..4651))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4666..4687
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4804..4821)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4975..5563)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5055..5074)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5734..6525,6526..6594))
                     /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     6357..6376
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6595..6699)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6774..6793)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6965..7344
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7345..7548
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7499..7519
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     7545..7569
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          join(7669..7971,1..270)
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
ORIGIN
        1 caaggcaatc agggtatatt atattgtact tcagcacagt tttagagaac aattgttata
       61 attaaatgat aaggtagaat atttctgcat ataaattctg gctggcgtgg aaatattctt
      121 attggtagaa acaactacat cctggtcatc atcctgcctt tctctttatg gttacaatga
      181 tatacactgt ttgagatgag gataaaatac tctgagtcca aaccgggccc ctctgctaac
      241 catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg
      301 tctcatcatt ttggcaaaga attgtaatac gactcactat agggcgaatt cggatccact
      361 agtccagtgt ggtggaattc tgcagatcta gagcctctgc taaccatgtt catgccttct
      421 tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca
      481 aagaattcga gctcatcgat gcatggtacc gccaccatgg ccaatagaag caggtatcag
      541 tcacgcaaag tggaaaatat gaaagcaaca atcatccaga gactaaagag aggctgtaaa
      601 ctgtatttaa tggcagagac cccgaaagtc ccgacttatc ggaccatata cccggcgcct
      661 gtgtactcgc ctccgatcaa tgtccgattg tccaaccccg agtccgcagt ggcagcatgt
      721 aatgagttct tagctagaaa ctacccaact gtttcatcat accaaatcac cgacgagtat
      781 gatgcatatc tagacatggt ggacgggtcg gagagttgct tggaccgagc gacattcaat
      841 ccgtcaaaac ttaggagcta cccgaaacaa catgcttatc acgcgccttc tatcagaagc
      901 gctgtacctt ccccattcca gaacacacta cagaatgtac tggcagcagc cacgaaaagg
      961 aactgcaacg tcacacagat gagggaatta cccactttgg actcagcagt attcaacgtg
     1021 gagtgtttta aaaaattcgc atgtaaccga gaatactggg aagaatttgc agccagccct
     1081 atcaggataa caactgagaa tctaacaacc tatgtcacta aactaaaggg gccaaaagca
     1141 gcagcgctgt ttgcaaaaac ccataatctg ctgccactgc aggatgtacc aatggatagg
     1201 ttcacagtag atatgaaaag ggatgtgaag gtaactcctg gtacaaagca tacagaggaa
     1261 agacctaagg tgcaggttat acaggcggct gaacccttgg caacagcgta cctatgtgga
     1321 attcacagag aactggttag gagattgaac gccgtcctcc tacccaatgt gcatacacta
     1381 tttgacatgt ctgccgagga cttcgatgcc attatagccg cacacttcaa gccaggagac
     1441 gctgttttag aaacggacat agcctccttt gataagagcc aagatgattc acttgcgctt
     1501 accgccttaa tgctgttaga agatttggga gtggatcact ccctgttgga cttgatagag
     1561 gctgctttcg gagagatttc cagctgtcat ctgccgacag gtacgcgctt caagttcggc
     1621 gctatgatga aatccggtat gttcctaact ctgttcgtca acacgttgtt aaatatcacc
     1681 atcgctagcc gggtgttgga agatcgtctg acaaaatccg catgcgcggc cttcatcggc
     1741 gacgacaaca taatacatgg tgtcgtctcc gatgaattga tggcagccag atgcgctact
     1801 tggatgaaca tggaagtgaa gatcatagat gcagttgtat cccagaaagc tccttacttt
     1861 tgtggagggt ttatactgca tgatactgtg acaggaacag cttgcagagt ggcggacccg
     1921 ctaaaaaggt tatttaaatt gggcaaaccg ttagcggcag gtgacgaaca agatgaagac
     1981 agaagacggg cgctggctga tgaagtaatc agatggcaac gaacagggct aatagatgag
     2041 ctggagaaag cggtgtactc taggtacgaa gtgcagggta tatcagttgc ggtaatgtcc
     2101 atggccacct ttgcaagctc cagatccaac ttcgagaagc tcagaggacc cgtcataact
     2161 ttgtacggcg gtcctaaaga ctacaaagac catgacggtg attataaaga tcatgatatc
     2221 gactacaagg atgacgatga caagtgactc gagctagcag atcatccagc acagtggcgg
     2281 ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt
     2341 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     2401 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     2461 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     2521 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     2581 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     2641 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     2701 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     2761 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     2821 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     2881 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     2941 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     3001 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
     3061 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3121 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     3181 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     3241 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     3301 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     3361 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat
     3421 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     3481 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     3541 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     3601 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     3661 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     3721 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     3781 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     3841 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     3901 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     3961 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     4021 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     4081 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     4141 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     4201 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     4261 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     4321 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     4381 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
     4441 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     4501 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
     4561 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     4621 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     4681 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4741 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     4801 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     4861 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     4921 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     4981 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5041 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5101 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5161 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     5221 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5281 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5341 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5401 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5461 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     5521 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     5581 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     5641 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     5701 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     5761 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     5821 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     5881 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     5941 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     6001 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     6061 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     6121 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     6181 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     6241 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     6301 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     6361 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     6421 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     6481 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     6541 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     6601 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     6661 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     6721 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc
     6781 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
     6841 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
     6901 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
     6961 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
     7021 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     7081 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     7141 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     7201 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     7261 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     7321 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
     7381 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     7441 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
     7501 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac
     7561 cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac
     7621 cgatccagcc tccgggggat cgatcccccc gatcctgaga acttcagggt gagtttgggg
     7681 acccttgatt gttctttctt tttcgctatt gtaaaattca tgttatatgg agggggcaaa
     7741 gttttcaggg tgttgtttag aatgggaaga tgtcccttgt atcaccatgg accctcatga
     7801 taattttgtt tctttcactt tctactctgt tgacaaccat tgtctcctct tattttcttt
     7861 tcattttctg taactttttc gttaaacttt agcttgcatt tgtaacgaat ttttaaattc
     7921 acttttgttt atttgtcaga ttgtaagtac tttctctaat cacttttttt t
//
