LOCUS       sequence-349861-        7225 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express gRNA expression cassette simultaneously targeting two 
            genes: UL8 and UL29 (non-EGFP version)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7225)
  AUTHORS   Yin D, Ling S, Wang D, Dai Y, Jiang H, Zhou X, Paludan SR, Hong J, 
            Cai Y
  TITLE     Targeting herpes simplex virus with CRISPR-Cas9 cures herpetic 
            stromal keratitis in mice.
  JOURNAL   Nat Biotechnol. 2021 Jan 11. pii: 10.1038/s41587-020-00781-8. doi: 
            10.1038/s41587-020-00781-8.
  PUBMED    33432198
REFERENCE   2  (bases 1 to 7225)
  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: "Nat 
            Biotechnol. 2021 Jan 11. pii: 10.1038/s41587-020-00781-8. doi: 
            10.1038/s41587-020-00781-8."
COMMENT     Sequence Label: pLV-U6-UL29-U3-UL8
FEATURES             Location/Qualifiers
     source          1..7225
                     /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"
     LTR             599..779
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    826..951
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1444..1677
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1862..1906
                     /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"
     misc_feature    2173..2290
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2317..2565
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_feature    2728..3316
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2781..2801)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3199..3210)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        3437..3685
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     LTR             3819..3999
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(4028..4046)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4029..4046)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4292..4311)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4293..4311)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4321..4338)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4321..4337)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4330..4352)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4479..4934
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4566..4585)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4776..4797
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4960..5064
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5065..5133,5134..5925)
                     /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(5283..5302)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6096..6684
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6585..6604
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6838..6855
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6972..6993
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7008..7013,7014..7031,7032..7038)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7046..7062
                     /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     7051..7073
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7070..7086
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7070..7086
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7105..7125
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7107..7125
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
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 agctcgttta gtgaaccggg
      601 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
      661 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
      721 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
      781 tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg
      841 actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca
      901 aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag
      961 cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa
     1021 tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct
     1081 ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt
     1141 cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg
     1201 catcaaagga tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa
     1261 aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga
     1321 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1381 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1441 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1501 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1561 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1621 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1681 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1741 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     1801 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     1861 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     1921 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     1981 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     2041 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga
     2101 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatctcgacg
     2161 gtatcggtta acttttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
     2221 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
     2281 tcaaaatttt atcgatcacg agactagcct cgacgagagg gcctatttcc catgattcct
     2341 tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta
     2401 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     2461 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     2521 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgcgag cgtacacgta
     2581 tcccgtttca gagctatgct ggaaacagca tagcaagttg aaataaggct agtccgttat
     2641 caacttgaaa aagtggcacc gagtcggtgc tttttttggt cgagtaccgg gtaggggagg
     2701 cgctagcggc ctcgagggaa ttccgataat caacctctgg attacaaaat ttgtgaaaga
     2761 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     2821 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     2881 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     2941 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     3001 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     3061 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     3121 aagctgacgt cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
     3181 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     3241 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     3301 tgggccgcct ccccgcatcg ggaattcgag ctcggtacct ttaagaccaa tgacttacaa
     3361 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca
     3421 ctcccaacga agacaagagg gcctatttcc catgattcct tcatatttgc atatacgata
     3481 caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca
     3541 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     3601 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     3661 tatcttgtgg aaaggacgaa acaccggggc agccataccg cgtaagtttc agagctatgc
     3721 tggaaacagc atagcaagtt gaaataaggc tagtccgtta tcaacttgaa aaagtggcac
     3781 cgagtcggtg cttttttgat ctgctttttg cttgtactgg gtctctctgg ttagaccaga
     3841 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
     3901 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     3961 ccctcagacc cttttagtca gtgtggaaaa tctctagcag catctagcta gaattaattc
     4021 cgtgtattct atagtgtcac ctaaatcgta tgtgtatgat acataaggtt atgtattaat
     4081 tgtagccgcg ttctaacgac aatatgtaca agcctaattg tgtagcatct ggcttactga
     4141 agcagaccct atcatctctc tcgtaaactg ccgtcagagt cggtttggtt ggacgaacct
     4201 tctgagtttc tggtaacgcc gtcccgcacc cggaaatggt cagcgaacca atcagcaggg
     4261 tcatcgctag cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc
     4321 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     4381 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     4441 cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt
     4501 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     4561 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     4621 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     4681 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     4741 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     4801 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     4861 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     4921 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     4981 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     5041 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     5101 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     5161 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     5221 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     5281 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     5341 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     5401 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     5461 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     5521 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     5581 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     5641 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     5701 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     5761 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     5821 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     5881 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     5941 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     6001 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     6061 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     6121 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     6181 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     6241 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     6301 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     6361 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     6421 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     6481 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     6541 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     6601 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     6661 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     6721 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     6781 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     6841 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     6901 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     6961 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     7021 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     7081 tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag
     7141 ctgcaagctt ggccattgca tacgttgtat ccatatcata atatgtacat ttatattggc
     7201 tcatgtccaa cattaccgcc atgtt
//
