LOCUS       sequence-382822-        8306 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral construct with mCherry and Puro cassettes to express 
            sgRNA-7 targeting the promoter of human MECP2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8306)
  AUTHORS   Qian J, Guan X, Xie B, Xu C, Niu J, Tang X, Li CH, Colecraft HM, 
            Jaenisch R, Liu XS
  TITLE     Multiplex epigenome editing of MECP2 to rescue Rett syndrome 
            neurons.
  JOURNAL   Sci Transl Med. 2023 Jan 18;15(679):eadd4666. doi: 
            10.1126/scitranslmed.add4666. Epub 2023 Jan 18.
  PUBMED    36652535
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"; journalName: "Sci Transl 
            Med."; date: "2023-01-18"; volume: "15(679):eadd4666. doi"; pages: "
            10.1126/scitranslmed.add4666. Epub 2023 Jan 18"
COMMENT     Sequence Label: pgRNA_hMECP2_promoter_No7
FEATURES             Location/Qualifiers
     source          1..8306
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        129..508
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        510..708
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             726..906
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    953..1078
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1575..1808
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1993..2037
                     /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    2335..2452
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2705..2738)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2742..2762
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     enhancer        3054..3357
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3358..3561
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3595..4191
                     /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(3595..3614)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4091..4111
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4201..4254
                     /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      4261..4270
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4267..4977
                     /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(4412..4430)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4680..4699
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(4997..5030)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5086..5674
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5139..5159)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5557..5568)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5677..5693)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5678..5694)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6203..6383
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6445..7033)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6525..6544)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7204..7995,7996..8064))
                     /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     7827..7846
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8065..8169)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8244..8263)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
       61 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
      121 tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
      181 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
      241 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
      301 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
      361 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
      421 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
      481 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
      541 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
      601 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
      661 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg cgttttgcct
      721 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
      781 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
      841 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
      901 ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg
      961 acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag
     1021 tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag
     1081 tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa
     1141 gaaaaaatat aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt
     1201 taatcctggc ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc
     1261 atcccttcag acaggatcag aagaacttag atcattatat aatacagtag caaccctcta
     1321 ttgtgtgcat caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga
     1381 agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc ggccgctgat cttcagacct
     1441 ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa
     1501 attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
     1561 agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg
     1621 ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag
     1681 cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc
     1741 tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa
     1801 cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg
     1861 aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag
     1921 tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa
     1981 aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg
     2041 aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga
     2101 ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag
     2161 ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc
     2221 gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac
     2281 ggatcggcac tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa
     2341 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
     2401 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta
     2461 ttacagggac agcagagatc cagtttggtt agtaccgggc ccgctctaga gatccgacgc
     2521 gccatctcta ggcccgcgcc ggccccctcg cacggacttg tgggagaagc tcggctactc
     2581 ccctgccccg gttaatttgc atataatatt tcctagtaac tatagaggct taatgtgcga
     2641 taaaagacag ataatctgtt ctttttaata ctagctacat tttacatgat aggcttggat
     2701 ttctataact tcgtatagca tacattatac gaagttataa acagcacaaa aggaaactca
     2761 ccctaactgt aaagtaattg tgtgttttga gactataagt atcccttgga gaaccacctt
     2821 gttgggggag gaagaggggc gtcgtttaag agctatgctg gaaacagcat agcaagttta
     2881 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttctcg
     2941 agtactagga tccattaggc ggccgcgtgg ataaccgtat taccgccatg cattagttat
     3001 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
     3061 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
     3121 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
     3181 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
     3241 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     3301 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
     3361 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
     3421 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
     3481 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
     3541 gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcc caccatgacc
     3601 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tcccccgggc cgtacgcacc
     3661 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgaccc ggaccgccac
     3721 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     3781 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     3841 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     3901 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     3961 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     4021 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
     4081 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     4141 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc cggatcggga
     4201 gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccaccggtc
     4261 gccaccatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc
     4321 ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc
     4381 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc
     4441 ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg
     4501 aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg
     4561 gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg
     4621 caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc
     4681 cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag
     4741 gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac
     4801 gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac
     4861 aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag
     4921 tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtaagaa
     4981 ttcgtcgagg gacctaataa cttcgtatag catacattat acgaagttat acatgtttaa
     5041 gggttccggt tccactaggt acaattcgat atcaagctta tcgataatca acctctggat
     5101 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     5161 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     5221 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     5281 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     5341 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     5401 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     5461 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
     5521 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     5581 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     5641 acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgtcgacc tcgatcgaga
     5701 cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc
     5761 ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt
     5821 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg
     5881 actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt ggatctacca
     5941 cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga tcagatatcc
     6001 actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg tagaagaagc
     6061 caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga tggatgaccc
     6121 ggagagagaa gtattagagt ggaggtttga cagccgccta gcatttcatc acatggcccg
     6181 agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag cctgggagct
     6241 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     6301 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     6361 gtcagtgtgg aaaatctcta gcagcatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     6421 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     6481 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     6541 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     6601 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     6661 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     6721 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     6781 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     6841 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     6901 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6961 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     7021 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     7081 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     7141 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     7201 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     7261 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     7321 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     7381 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     7441 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     7501 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     7561 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     7621 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     7681 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     7741 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     7801 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     7861 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     7921 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     7981 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     8041 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     8101 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     8161 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc
     8221 gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     8281 ctgctccctg cttgtgtgtt ggaggt
//
