LOCUS       sequence-224459-        5727 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCMV-Tat101AD8-Flag allows the expression of Tat101(AD8) protein 
            with a C-terminal flag-tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5727)
  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 5727)
  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: pcDNA3.1-Tat101(AD8)-Flag
FEATURES             Location/Qualifiers
     source          1..5727
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1224..1247
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1318..1335)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1324..1548
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1594..2022
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1681..1700)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1891..1912
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2031..2051)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2036..2365
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2216..2351
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2278..2297
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2432..3226
                     /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(2486..2505)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3096..3115
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3400..3521
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3437..3456)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3491..3510
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3570..3586)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3570..3586)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3583..3605)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3594..3610
                     /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(3618..3624,3625..3642,3643..3648))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3663..3684
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3801..3818)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3972..4560)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4052..4071)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4731..5522,5523..5591))
                     /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     5354..5373
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5592..5696)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaacgg gccctctaga atggagccag tagatcctag actagagccc tggaagcatc
      961 caggaagcca gcctaggact gcttgtaaca attgctattg taaaaagtgt tgctttcatt
     1021 gccaagtttg cttcacaaga aaaggcttag gcatctccta tggcaggaag aagcggagac
     1081 agcgacgaag agctcctcaa gacagtcaga ctcatcaagt ttctctatca aagcaaccca
     1141 cctcccagcc ccgaggggac ccgacaggcc cgaaggaatc gaagaagaag gtggagacag
     1201 agacagagac agatccgtgc gatgattata aagatgatga tgataaatag gaattccacc
     1261 acactggact agtggatccg agctcggtac caagcttaag tttaaaccgc tgatcagcct
     1321 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     1381 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     1441 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     1501 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     1561 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     1621 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     1681 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     1741 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1801 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1861 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1921 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1981 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
     2041 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2101 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2161 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     2221 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     2281 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     2341 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
     2401 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     2461 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     2521 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     2581 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     2641 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     2701 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     2761 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     2821 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     2881 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     2941 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     3001 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     3061 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     3121 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     3181 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     3241 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     3301 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     3361 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     3421 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     3481 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     3541 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     3601 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     3661 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3721 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3781 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3841 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3901 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3961 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     4021 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     4081 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     4141 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     4201 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     4261 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     4321 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     4381 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     4441 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4501 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4561 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4621 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4681 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     4741 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     4801 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     4861 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     4921 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     4981 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     5041 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     5101 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     5161 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     5221 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     5281 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     5341 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     5401 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     5461 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     5521 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     5581 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     5641 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     5701 tttccccgaa aagtgccacc tgacgtc
//
