LOCUS       sequence-256928-        8447 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector for expression of KSHV ORF66 with a C-terminal 
            2xStrep tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8447)
  AUTHORS   Didychuk AL, Castaneda AF, Kushnir LO, Huang CJ, Glaunsinger BA
  TITLE     Conserved CxnC motifs in Kaposi's sarcoma-associated herpesvirus 
            ORF66 are required for viral late gene expression and are essential 
            for its interaction with ORF34.
  JOURNAL   J Virol. 2019 Oct 2. pii: JVI.01299-19. doi: 10.1128/JVI.01299-19.
  PUBMED    31578296
REFERENCE   2  (bases 1 to 8447)
  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. 
            2019 Oct 2. pii: JVI.01299-19. doi: 10.1128/JVI.01299-19."
COMMENT     Sequence Label: pLJM1-zeo-ORF66-2xStrep
FEATURES             Location/Qualifiers
     source          1..8447
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        131..334
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     285..305
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1688..1711
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1748..1771
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     misc_feature    1824..1941
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1990..2500
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2419..2437
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2509..2883
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     LTR             2955..3188
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3260..3381
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3297..3316)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3351..3370
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3421..3556
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3576..3595)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3577..3595)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3605..3622)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3605..3621)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3614..3636)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3763..4218
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3850..3869)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4060..4081
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4244..4348
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4349..4417,4418..5209)
                     /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(4567..4586)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5380..5968
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5869..5888
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6122..6139
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6256..6277
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6292..6297,6298..6315,6316..6322)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6330..6346
                     /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)."
     primer_bind     6335..6357
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6354..6370
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6354..6370
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6389..6409
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6391..6409
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6437..6663
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6664..6844
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6891..7016
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7509..7742
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7927..7971
                     /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"
     primer_bind     8269..8289
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
       61 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      121 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      181 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      241 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      301 gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta
      361 gcgctaccgg atggccctgg atcagcgctg ggatcgcttt cttgtgtctt ggtttggttt
      421 ggatgaggcc cagctgacgg cccaccgcgt gttcgagggc gagaatggcg tgccagtgga
      481 ggaatatgtg gcttttgtca tattcgggga gcggggtttc cagggaaaca tgccctcctg
      541 ggccaggcac ctgttggacc gcccgtcgct ggcgcaggcg atcgccgtgt tgcgcgctgg
      601 ctcagacacc gtcgccaaac aggcgcagat ttgtgcggcg cagcagctgc ttggcgccca
      661 cgtatgggtt gtggtgacgc tgtcccgtgc ccaggccgcc gaccatgcta gggcgatacc
      721 gcgccacgta tgggcaaaat acctgtcgct tccattttcc aaggcctgtg cccaactctg
      781 caagctgttg gcgctttgct ctcgctttcc gttagtgacc tgctgttcga agcccccacc
      841 gtcgctaccc tggctaagga agaagtggca tgggccgttg ccgcgtcgtc ccctcctgga
      901 ggtgccctcg cccacccgtc gcggtgtggc tgcgaccgga gacggtaacg ggctcgggat
      961 aggggcggca gataccggcc tccgggaggc cttggagcgc gtcgccccta cggttccctg
     1021 cggtaacccg ttcgacgcca tgcttgggtc actgtgtttt ttgagcctta ttaagtcccg
     1081 ccacgtagtg ctgcccgcct gcgagcagga gggacctggt ctcgtgcgga accttggcag
     1141 gaggcttctg gcatacaatg ttctctcccc ctgcgtgtcc atacccgtca tctgttcgcg
     1201 cgtggcccgc gcggcccttg ccaagcgggc gcgttgcgct cgggcggttg tgtgcatgga
     1261 gtgtggacac tgtcttaact ttggcagggg caagtttcat actgtcaatt ttcctcccac
     1321 caacgtgttt ttcagcaggg acaggaaaga gaagcagttc accatctgtg cgaccacggg
     1381 gaggatctac tgttcttact gtggcagcga acatatgagg gtgtatcccc tgtgcgatat
     1441 taccggacgc gggaccctag cacgcgtcgt aatcagggct gtcctagcta acaacgcggc
     1501 ccttgccatt cgagacctag atcaaactgt cagttttgta gtgccttgcc ttgggacgcc
     1561 cgactgcgag gctgccctat taaagcaccg tgacgtgcgc ggactgcttc agctcacctc
     1621 acagctgctg gagttctgtt gcgggaagtg ttcctccgga gggcggccgc tcgagggagg
     1681 cggtggatgg agccaccccc agttcgagaa gggcggcggc agcggcggcg gcagcggcgg
     1741 cggcagctgg agccaccccc agttcgagaa ggggcccgtt taaaattctc gacctcgaga
     1801 caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca
     1861 ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa
     1921 attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc actttggccg
     1981 cggctcgagg gggttggggt tgcgcctttt ccaaggcagc cctgggtttg cgcagggacg
     2041 cggctgctct gggcgtggtt ccgggaaacg cagcggcgcc gaccctgggt ctcgcacatt
     2101 cttcacgtcc gttcgcagcg tcacccggat cttcgccgct acccttgtgg gccccccggc
     2161 gacgcttcct gctccgcccc taagtcggga aggttccttg cggttcgcgg cgtgccggac
     2221 gtgacaaacg gaagccgcac gtctcactag taccctcgca gacggacagc gccagggagc
     2281 aatggcagcg cgccgaccgc gatgggctgt ggccaatagc ggctgctcag cagggcgcgc
     2341 cgagagcagc ggccgggaag gggcggtgcg ggaggcgggg tgtggggcgg tagtgtgggc
     2401 cctgttcctg cccgcgcggt gttccgcatt ctgcaagcct ccggagcgca cgtcggcagt
     2461 cggctccctc gttgaccgaa tcaccgacct ctctccccag ggggatccat ggccaagttg
     2521 accagtgccg ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc
     2581 gaccggctcg ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac
     2641 gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc
     2701 tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg
     2761 aacttccggg acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg
     2821 gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga ggagcaggac
     2881 tgaggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
     2941 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc
     3001 ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     3061 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     3121 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     3181 ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg
     3241 aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa
     3301 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3361 caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc
     3421 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3481 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     3541 ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc
     3601 gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     3661 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     3721 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
     3781 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     3841 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     3901 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     3961 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     4021 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     4081 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     4141 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     4201 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     4261 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     4321 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     4381 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     4441 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     4501 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     4561 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     4621 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     4681 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     4741 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     4801 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     4861 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     4921 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     4981 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     5041 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     5101 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     5161 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     5221 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     5281 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     5341 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     5401 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     5461 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     5521 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     5581 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5641 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5701 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5761 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     5821 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     5881 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5941 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     6001 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     6061 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     6121 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     6181 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     6241 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     6301 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     6361 cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct
     6421 ggagctgcaa gcttaatgta gtcttatgca atactcttgt agtcttgcaa catggtaacg
     6481 atgagttagc aacatgcctt acaaggagag aaaaagcacc gtgcatgccg attggtggaa
     6541 gtaaggtggt acgatcgtgc cttattagga aggcaacaga cgggtctgac atggattgga
     6601 cgaaccactg aattgccgca ttgcagagat attgtattta agtgcctagc tcgatacata
     6661 aacgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     6721 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     6781 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     6841 agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac
     6901 gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta
     6961 cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta
     7021 ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga
     7081 aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta
     7141 atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat
     7201 cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca accctctatt
     7261 gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag
     7321 agcaaaacaa aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga
     7381 ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa
     7441 ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca
     7501 gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca
     7561 gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag
     7621 aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc
     7681 atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc
     7741 ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct
     7801 agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac
     7861 agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag
     7921 caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg
     7981 tttaacataa caaattggct gtggtatata aaattattca taatgatagt aggaggcttg
     8041 gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat
     8101 tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga
     8161 atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatct
     8221 cgacggtatc gatcacgaga ctagcctcga gcggccgccc ccttcaccga gggcctattt
     8281 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt
     8341 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
     8401 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatca
//
