LOCUS       sequence-306301-        8306 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  guide RNA expression lentiviral construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8306)
  AUTHORS   Liu XS, Wu H, Ji X, Stelzer Y, Wu X, Czauderna S, Shu J, Dadon D, 
            Young RA, Jaenisch R
  TITLE     Editing DNA Methylation in the Mammalian Genome.
  JOURNAL   Cell. 2016 Sep 22;167(1):233-247.e17. doi: 
            10.1016/j.cell.2016.08.056.
  PUBMED    27662091
REFERENCE   2  (bases 1 to 8306)
  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"; doi: 
            "10.1016/j.cell.2016.08"; journalName: "Cell"; date: "2016-09-22- 
            22"; volume: "167"; issue: "1"; pages: "233-247.e17"
COMMENT     Sequence Label: pgRNA-modified
FEATURES             Location/Qualifiers
     source          1..8306
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..580
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             598..778
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    825..950
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1447..1680
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1865..1909
                     /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    2207..2324
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2577..2610)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2614..2634
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        2726..2801
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        2926..3229
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3230..3433
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3467..4063
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3467..3486)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3963..3983
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4073..4126
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     regulatory      4133..4142
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4139..4849
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4284..4302)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4552..4571
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(4869..4902)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4958..5546
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5011..5031)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5429..5440)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5549..5565)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5550..5566)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6075..6255
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6317..6905)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6397..6416)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7076..7867,7868..7936))
                     /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     7699..7718
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7937..8041)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8116..8135)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
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 cgcgttttgc ctgtactggg
      601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
      781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
      841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
      901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
      961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     1261 acaaaagtaa gaccaccgca cagcaagcgg ccggccgctg atcttcagac ctggaggagg
     1321 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
     1381 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
     1441 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
     1501 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
     1561 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
     1621 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
     1681 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     1741 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     1801 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
     1861 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
     1921 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
     1981 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
     2041 accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat
     2101 agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatcggc
     2161 actgcgtgcg ccaattctgc agacaaatgg cagtattcat ccacaatttt aaaagaaaag
     2221 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     2281 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     2341 acagcagaga tccagtttgg ttagtaccgg gcccgctcta gagatccgac gcgccatctc
     2401 taggcccgcg ccggccccct cgcacggact tgtgggagaa gctcggctac tcccctgccc
     2461 cggttaattt gcatataata tttcctagta actatagagg cttaatgtgc gataaaagac
     2521 agataatctg ttctttttaa tactagctac attttacatg ataggcttgg atttctataa
     2581 cttcgtatag catacattat acgaagttat aaacagcaca aaaggaaact caccctaact
     2641 gtaaagtaat tgtgtgtttt gagactataa gtatcccttg gagaaccacc ttgttggtct
     2701 cgcaggtggg atcccacctg ctctcgtttt agagctagaa atagcaagtt aaaataaggc
     2761 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttct cgagtactag
     2821 gatccattag gcggccgcgt ggataaccgt attaccgcca tgcattagtt attaatagta
     2881 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     2941 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     3001 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     3061 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
     3121 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     3181 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
     3241 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     3301 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     3361 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     3421 tataagcaga gctggtttag tgaaccgtca gatccgctag cccaccatga ccgagtacaa
     3481 gcccacggtg cgcctcgcca cccgcgacga cgtcccccgg gccgtacgca ccctcgccgc
     3541 cgcgttcgcc gactaccccg ccacgcgcca caccgtcgac ccggaccgcc acatcgagcg
     3601 ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg
     3661 ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg
     3721 ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc
     3781 gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct
     3841 ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct
     3901 ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc
     3961 ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc
     4021 cgaaggaccg cgcacctggt gcatgacccg caagcccggt gccggatcgg gagagggcag
     4081 aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccaccgg tcgccaccat
     4141 ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt
     4201 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
     4261 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt
     4321 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
     4381 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     4441 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     4501 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
     4561 gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc
     4621 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga
     4681 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa
     4741 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg
     4801 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtaag aattcgtcga
     4861 gggacctaat aacttcgtat agcatacatt atacgaagtt atacatgttt aagggttccg
     4921 gttccactag gtacaattcg atatcaagct tatcgataat caacctctgg attacaaaat
     4981 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     5041 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     5101 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     5161 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     5221 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     5281 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     5341 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
     5401 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     5461 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     5521 gatctccctt tgggccgcct ccccgcatcg ataccgtcga cctcgatcga gacctagaaa
     5581 aacatggagc aatcacaagt agcaatacag cagctaccaa tgctgattgt gcctggctag
     5641 aagcacaaga ggaggaggag gtgggttttc cagtcacacc tcaggtacct ttaagaccaa
     5701 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag
     5761 ggctaattca ctcccaacga agacaagata tccttgatct gtggatctac cacacacaag
     5821 gctacttccc tgattggcag aactacacac cagggccagg gatcagatat ccactgacct
     5881 ttggatggtg ctacaagcta gtaccagttg agcaagagaa ggtagaagaa gccaatgaag
     5941 gagagaacac ccgcttgtta caccctgtga gcctgcatgg gatggatgac ccggagagag
     6001 aagtattaga gtggaggttt gacagccgcc tagcatttca tcacatggcc cgagagctgc
     6061 atccggactg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct
     6121 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
     6181 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
     6241 ggaaaatctc tagcagcatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     6301 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     6361 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     6421 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     6481 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     6541 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     6601 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     6661 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     6721 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     6781 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6841 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6901 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6961 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     7021 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     7081 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     7141 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     7201 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     7261 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     7321 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     7381 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     7441 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     7501 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     7561 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     7621 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     7681 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     7741 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     7801 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7861 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7921 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7981 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     8041 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     8101 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     8161 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
     8221 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
     8281 cgatgtacgg gccagatata cgcgtt
//
