LOCUS       sequence-382828-        8306 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral construct with mCherry and Puro cassettes to express 
            sgRNA-4 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_No4
FEATURES             Location/Qualifiers
     source          1..8306
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        167..546
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        548..746
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             764..944
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    991..1116
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1613..1846
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2031..2075
                     /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    2373..2490
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2743..2776)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2780..2800
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     enhancer        3092..3395
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3396..3599
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3633..4229
                     /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(3633..3652)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4129..4149
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4239..4292
                     /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      4299..4308
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4305..5015
                     /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(4450..4468)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4718..4737
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(5035..5068)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5124..5712
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5177..5197)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5595..5606)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5715..5731)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5716..5732)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6241..6421
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6483..7071)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6563..6582)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7242..8033,8034..8102))
                     /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     7865..7884
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8103..8207)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8282..8301)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
       61 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt
      121 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg
      181 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
      241 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      301 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      361 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      421 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      481 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      541 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      601 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      661 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      721 gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc tctggttaga
      781 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
      841 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
      901 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg
      961 gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa
     1021 gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc
     1081 ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga
     1141 tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat
     1201 atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca
     1261 tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa
     1321 gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag
     1381 ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc
     1441 accgcacagc aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca
     1501 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     1561 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     1621 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     1681 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     1741 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     1801 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     1861 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     1921 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     1981 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     2041 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     2101 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     2161 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     2221 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg
     2281 gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg cgtgcgccaa
     2341 ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg
     2401 tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta
     2461 caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca
     2521 gtttggttag taccgggccc gctctagaga tccgacgcgc catctctagg cccgcgccgg
     2581 ccccctcgca cggacttgtg ggagaagctc ggctactccc ctgccccggt taatttgcat
     2641 ataatatttc ctagtaacta tagaggctta atgtgcgata aaagacagat aatctgttct
     2701 ttttaatact agctacattt tacatgatag gcttggattt ctataacttc gtatagcata
     2761 cattatacga agttataaac agcacaaaag gaaactcacc ctaactgtaa agtaattgtg
     2821 tgttttgaga ctataagtat cccttggaga accaccttgt tgggccatca cagccaatga
     2881 cgtttaagag ctatgctgga aacagcatag caagtttaaa taaggctagt ccgttatcaa
     2941 cttgaaaaag tggcaccgag tcggtgcttt ttttctcgag tactaggatc cattaggcgg
     3001 ccgcgtggat aaccgtatta ccgccatgca ttagttatta atagtaatca attacggggt
     3061 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     3121 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     3181 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     3241 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     3301 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     3361 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     3421 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     3481 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     3541 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg
     3601 gtttagtgaa ccgtcagatc cgctagccca ccatgaccga gtacaagccc acggtgcgcc
     3661 tcgccacccg cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg ttcgccgact
     3721 accccgccac gcgccacacc gtcgacccgg accgccacat cgagcgggtc accgagctgc
     3781 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     3841 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     3901 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     3961 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     4021 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     4081 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     4141 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     4201 cctggtgcat gacccgcaag cccggtgccg gatcgggaga gggcagagga agtctgctaa
     4261 catgcggtga cgtcgaggag aatcctggcc caccggtcgc caccatggtg agcaagggcg
     4321 aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct
     4381 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
     4441 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc
     4501 tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg
     4561 actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
     4621 acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca
     4681 aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca
     4741 tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga
     4801 tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct
     4861 acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca
     4921 tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc
     4981 actccaccgg cggcatggac gagctgtaca agtaagaatt cgtcgaggga cctaataact
     5041 tcgtatagca tacattatac gaagttatac atgtttaagg gttccggttc cactaggtac
     5101 aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga
     5161 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     5221 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     5281 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     5341 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     5401 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     5461 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     5521 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     5581 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     5641 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     5701 ccgcctcccc gcatcgatac cgtcgacctc gatcgagacc tagaaaaaca tggagcaatc
     5761 acaagtagca atacagcagc taccaatgct gattgtgcct ggctagaagc acaagaggag
     5821 gaggaggtgg gttttccagt cacacctcag gtacctttaa gaccaatgac ttacaaggca
     5881 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc
     5941 caacgaagac aagatatcct tgatctgtgg atctaccaca cacaaggcta cttccctgat
     6001 tggcagaact acacaccagg gccagggatc agatatccac tgacctttgg atggtgctac
     6061 aagctagtac cagttgagca agagaaggta gaagaagcca atgaaggaga gaacacccgc
     6121 ttgttacacc ctgtgagcct gcatgggatg gatgacccgg agagagaagt attagagtgg
     6181 aggtttgaca gccgcctagc atttcatcac atggcccgag agctgcatcc ggactgtact
     6241 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
     6301 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
     6361 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
     6421 agcatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     6481 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     6541 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     6601 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     6661 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     6721 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     6781 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     6841 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     6901 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     6961 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     7021 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     7081 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     7141 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     7201 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     7261 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     7321 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     7381 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     7441 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     7501 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     7561 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     7621 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     7681 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     7741 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     7801 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     7861 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     7921 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     7981 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     8041 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     8101 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     8161 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     8221 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     8281 gtacaatctg ctctgatgcc gcatag
//
