LOCUS       sequence-224438-        8281 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HIV splicing reporter allows the assessment of the effect of LRAs on
            HIV-1 transcription and splicing. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8281)
  AUTHORS   Khoury G, Purcell DFJ
  TITLE     High Throughput In Vitro Assessment of Latency Reversing Agents on 
            HIV Transcription and Splicing.
  JOURNAL   J Vis Exp. 2019 Jan 22;(143). doi: 10.3791/58753.
  PUBMED    30735158
REFERENCE   2  (bases 1 to 8281)
  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 Vis 
            Exp."; date: "2019-01-22"; volume: "(143). doi"; pages: " 
            10.3791/58753"
COMMENT     Sequence Label: pLTR.gp140/EGFP.Revdelta38/DsRed
FEATURES             Location/Qualifiers
     source          1..8281
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             377..634
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2543..2776
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2961..3005
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     CDS             join(3057..3059,3060..3773)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(3057..3059,3060..3770)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3099..3120)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3360..3379)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3707..3728
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3926..4600
                     /codon_start=1
                     /product="rapidly maturing tetrameric variant of DsRed 
                     fluorescent protein (Bevis and Glick, 2002)"
                     /label=DsRed-Express
                     /note="mammalian codon-optimized"
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHKA
                     LKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHHL
                     FL"
     primer_bind     complement(4053..4071)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(4103..4123)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     4321..4340
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4515..4538
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             4709..4735
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     LTR             5270..5903
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(5953..5972)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6072..6094
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6132..6150)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6218..6322
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6323..6391,6392..7183)
                     /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     complement(6541..6560)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7354..7942
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7843..7862
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8096..8113
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tggaagggct aatttggtcc caaaaaagac aagagatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tggcagaact acacaccagg gccagggatc agatatccac
      121 tgacctttgg atggtgcttc aagttagtac cagttgaacc agagcaagta gaagaggcca
      181 atgaaggaga gaacaacagc ttgttacacc ctatgagcca gcatgggatg gaggacccgg
      241 agggagaagt attagtgtgg aagtttgaca gcctcctagc atttcgtcac atggcccgag
      301 agctgcatcc ggagtactac aaagactgct gacatcgagc tttctacaag ggactttccg
      361 ctggggactt tccagggagg tgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 gctacatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgctca aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagtaaa gccagaggag atctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggcatctc ctatggcagg aagaagcgga gacagcgacg
      781 aagagctcat cagaacagtc agactcatca agcttctcta tcaaagcagt aagtagtaca
      841 tgtaatgcaa cctataatag tagcaatagt agcattagta gtagcaataa taatagcaat
      901 agttgtgtgg tccatagtaa tcatagaata taggaaaata ttaagacaaa gaaaaataga
      961 caggttaatt gatagactaa tagaaagagc agaagacagt ggcaatgaga gtgaaggaga
     1021 agtatcagca cttgtggaga tgggggtgga aatggggcac catgctcctt gggatattga
     1081 tgatctgtag tgctacagaa aaattgtggg tcacagtcta ttatggggta cctgtgtgga
     1141 aggaagcaac caccactcta ttttgtgcat cagatgctaa agcatatgat acagaggtac
     1201 ataatgtttg ggccacacat gcctgtgtac ccacagaccc caacccacaa gaagtagtat
     1261 tggtaaatgt gacagaaaat tttaacatgt ggaaaaatga catggtagaa cagatgcatg
     1321 aggatataat cagtttatgg gatcaaagcc taaagccatg tgtaaaatta accccactct
     1381 gtgttagttt aaagtgcact gatttgaaga atgatactaa taccaatagt agtagcggga
     1441 gaatgataat ggagaaagga gagataaaaa actgctcttt caatatcagc acaagcataa
     1501 gagataaggt gcagaaagaa tatgcattct tttataaact tgatatagta ccaatagata
     1561 ataccagcta taggttgata agttgtaaca cctcagtcat tacacaggcc tgtccaaagg
     1621 tatcctttga gccaattccc atacattatt gtgccccggc tggttttgcg attctaaaat
     1681 gtaataataa gacgttcaat ggaacaggac catgtacaaa tgtcagcaca gtacaatgta
     1741 cacatggaat caggccagta gtatcaactc aactgctgtt aaatggcagt ctagcagaag
     1801 aagatgtagt aattagatct gccaatttca cagacaatgc taaaaccata atagtacagc
     1861 tgaacacatc tgtagaaatt aattgtacaa gacccaacaa caatacaaga aaaagtatcc
     1921 gtatccagag gggaccaggg agagcatttg ttacaatagg aaaaatagga aatatgagac
     1981 aagcacattg taacattagt agagcaaaat ggaatgccac tttaaaacag atagctagca
     2041 aattaagaga acaatttgga aataataaaa caataatctt taagcaatcc tcaggagggg
     2101 acccagaaat tgtaacgcac agttttaatt gtggagggga atttttctac tgtaattcaa
     2161 cacaactgtt taatagtact tggtttaata gtacttggag tactgaaggg tcaaataaca
     2221 ctgaaggaag tgacacaatc acactcccat gcagaataaa acaatttata aacatgtggc
     2281 aggaagtagg aaaagcaatg tatgcccctc ccatcagtgg acaaattaga tgttcatcaa
     2341 atattactgg gctgctatta acaagagatg gtggtaataa caacaatggg tccgagatct
     2401 tcagacctgg aggaggcgat atgagggaca attggagaag tgaattatat aaatataaag
     2461 tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga
     2521 gagaaaccgg tgcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa
     2581 gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctgata
     2641 tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac
     2701 tcacagtctg gggcatcaaa cagctccagg caagaatcct ggctgtggaa agatacctaa
     2761 aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg
     2821 tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat aacatgacct
     2881 ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag
     2941 aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa
     3001 gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaagct agcacggtga
     3061 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     3121 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     3181 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     3241 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     3301 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     3361 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     3421 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     3481 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     3541 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     3601 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     3661 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     3721 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag taaagcggcc
     3781 gcgactctag ctaaatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac
     3841 tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc
     3901 caatcccgag gggacccgac aggccgcctc ctccgaggac gtcatcaagg agttcatgcg
     3961 cttcaaggtg cgcatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg
     4021 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggcggccc
     4081 cctgcccttc gcctgggaca tcctgtcccc ccagttccag tacggctcca aggtgtacgt
     4141 gaagcacccc gccgacatcc ccgactacaa gaagctgtcc ttccccgagg gcttcaagtg
     4201 ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct
     4261 gcaggacggc tccttcatct acaaggtgaa gttcatcggc gtgaacttcc cctccgacgg
     4321 ccccgtaatg cagaagaaga ctatgggctg ggaggcctcc accgagcgcc tgtacccccg
     4381 cgacggcgtg ctgaagggcg agatccacaa ggccctgaag ctgaaggacg gcggccacta
     4441 cctggtggag ttcaagtcca tctacatggc caagaagccc gtgcagctgc ccggctacta
     4501 ctacgtggac tccaagctgg acatcacctc ccacaacgag gactacacca tcgtggagca
     4561 gtacgagcgc gccgagggcc gccaccacct gttcctgtga aggaatagaa gaagaaggtg
     4621 gagagagaga cagagacaga tccattcgat tagtgaacgg atccttagca cttatctggg
     4681 acgatctgcg gagcctgtgc ctcttcagct accaccactt gagagactta ctcttgattg
     4741 taacgaggat tgtggaactt ctgggacgca gggggtggga agccctcaaa tattggtgga
     4801 atctcctaca gtattggagt caggaactaa agaatagtgc tgttaacttg ctcaatgcca
     4861 cagccatagc agtagctgag gggacagata gggttataga agtattacaa gcagcttata
     4921 gagctattcg ccacatacct agaagaataa gacagggctt ggaaaggatt ttgctataag
     4981 atgggtggca agtggtcaaa aagtagtgtg attggatggc ctgctgtaag ggaaagaatg
     5041 agacgagctg agccagcagc agatggggtg ggagcagtat ctcgagacct agaaaaacat
     5101 ggagcaatca caagtagcaa tacagcagct aacaatgctg cttgtgcctg gctagaagca
     5161 caagaggagg aagaggtggg ttttccagtc acacctcagg tacctttaag accaatgact
     5221 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta
     5281 attcactccc aaagaagaca agatatcctt gatctgtgga tctaccacac acaaggctac
     5341 ttccctgatt ggcagaacta cacaccaggg ccaggggtca gatatccact gacctttgga
     5401 tggtgctaca agctagtacc agttgagcca gataaggtag aagaggccaa taaaggagag
     5461 aacaccagct tgttacaccc tgtgagcctg catggaatgg atgaccctga gagagaagtg
     5521 ttagagtgga ggtttgacag ccgcctagca tttcatcacg tggcccgaga gctgcatccg
     5581 gagtacttca agaactgctg acatcgagct tgctacaagg gactttccgc tggggacttt
     5641 ccagggaggc gtggcctggg cgggactggg gagtggcgag ccctcagatg ctgcatataa
     5701 gcagctgctt tttgcctgta ctgggtctct ctggttagac cagatctgag cctgggagct
     5761 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     5821 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     5881 gtcagtgtgg aaaatctcta gcacccagga ggtagaggtt gcagtgagcc aagatccatg
     5941 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     6001 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     6061 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     6121 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     6181 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     6241 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     6301 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     6361 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     6421 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     6481 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     6541 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     6601 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     6661 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     6721 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     6781 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     6841 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     6901 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     6961 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     7021 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     7081 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     7141 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     7201 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     7261 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     7321 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     7381 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     7441 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     7501 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     7561 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     7621 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     7681 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     7741 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     7801 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     7861 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     7921 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     7981 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     8041 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     8101 gtcagtgagc gaggatggtg accttcgtgc acatggccgg aggaactgcc atgtcggagg
     8161 tgcaagcaca cctgcgcatc agagtccttg gtgtggaggg agggaccagc gcagcttcca
     8221 gccatccacc tgatgaacag aacctaggga aagccccagt tctacttaca ccaggaaagg
     8281 c
//
