LOCUS       sequence-382876-        8308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral construct with mCherry and Puro cassettes to express 
            sgRNA-3 targeting the promoter of human MECP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8308)
  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 8308)
  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_No3
FEATURES             Location/Qualifiers
     source          1..8308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        183..562
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        564..762
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             780..960
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1007..1132
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1629..1862
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2047..2091
                     /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    2389..2506
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2759..2792)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2796..2816
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     enhancer        3110..3413
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3414..3617
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3651..4247
                     /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(3651..3670)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4147..4167
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4257..4310
                     /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      4317..4326
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4323..5033
                     /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(4468..4486)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4736..4755
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(5053..5086)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5142..5730
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5195..5215)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5613..5624)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5733..5749)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5734..5750)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6259..6439
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6501..7089)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6581..6600)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7260..8051,8052..8120))
                     /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     7883..7902
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8121..8225)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(join(8300..8308,1..11))
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
       61 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
      121 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
      181 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
      241 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      301 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      361 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      421 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      481 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      541 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      601 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      661 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      721 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agcgcgtttt gcctgtactg
      781 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
      841 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
      901 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
      961 gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
     1021 gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
     1081 aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa
     1141 gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
     1201 atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc
     1261 tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct
     1321 tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt
     1381 gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca
     1441 aaacaaaagt aagaccaccg cacagcaagc ggccggccgc tgatcttcag acctggagga
     1501 ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa
     1561 ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca
     1621 gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca
     1681 gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag
     1741 aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc
     1801 atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc
     1861 ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct
     1921 agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac
     1981 agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag
     2041 caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg
     2101 tttaacataa caaattggct gtggtatata aaattattca taatgatagt aggaggcttg
     2161 gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat
     2221 tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga
     2281 atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatcg
     2341 gcactgcgtg cgccaattct gcagacaaat ggcagtattc atccacaatt ttaaaagaaa
     2401 aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat
     2461 acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag
     2521 ggacagcaga gatccagttt ggttagtacc gggcccgctc tagagatccg acgcgccatc
     2581 tctaggcccg cgccggcccc ctcgcacgga cttgtgggag aagctcggct actcccctgc
     2641 cccggttaat ttgcatataa tatttcctag taactataga ggcttaatgt gcgataaaag
     2701 acagataatc tgttcttttt aatactagct acattttaca tgataggctt ggatttctat
     2761 aacttcgtat agcatacatt atacgaagtt ataaacagca caaaaggaaa ctcaccctaa
     2821 ctgtaaagta attgtgtgtt ttgagactat aagtatccct tggagaacca ccttgttggc
     2881 agccaatcaa cagctggagg tttaagagct atgctggaaa cagcatagca agtttaaata
     2941 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttctcgagta
     3001 ctaggatcca ttaggcggcc gcgtggataa ccgtattacc gccatgcatt agttattaat
     3061 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
     3121 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
     3181 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
     3241 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
     3301 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
     3361 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
     3421 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
     3481 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
     3541 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
     3601 tctatataag cagagctggt ttagtgaacc gtcagatccg ctagcccacc atgaccgagt
     3661 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc ccgggccgta cgcaccctcg
     3721 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgacccggac cgccacatcg
     3781 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     3841 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     3901 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     3961 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     4021 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     4081 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     4141 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     4201 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgccgga tcgggagagg
     4261 gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa tcctggccca ccggtcgcca
     4321 ccatggtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca
     4381 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
     4441 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
     4501 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc
     4561 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc
     4621 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg
     4681 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg
     4741 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg
     4801 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg
     4861 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg
     4921 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg
     4981 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag taagaattcg
     5041 tcgagggacc taataacttc gtatagcata cattatacga agttatacat gtttaagggt
     5101 tccggttcca ctaggtacaa ttcgatatca agcttatcga taatcaacct ctggattaca
     5161 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     5221 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     5281 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     5341 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     5401 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     5461 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     5521 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga
     5581 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     5641 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     5701 gtcggatctc cctttgggcc gcctccccgc atcgataccg tcgacctcga tcgagaccta
     5761 gaaaaacatg gagcaatcac aagtagcaat acagcagcta ccaatgctga ttgtgcctgg
     5821 ctagaagcac aagaggagga ggaggtgggt tttccagtca cacctcaggt acctttaaga
     5881 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
     5941 gaagggctaa ttcactccca acgaagacaa gatatccttg atctgtggat ctaccacaca
     6001 caaggctact tccctgattg gcagaactac acaccagggc cagggatcag atatccactg
     6061 acctttggat ggtgctacaa gctagtacca gttgagcaag agaaggtaga agaagccaat
     6121 gaaggagaga acacccgctt gttacaccct gtgagcctgc atgggatgga tgacccggag
     6181 agagaagtat tagagtggag gtttgacagc cgcctagcat ttcatcacat ggcccgagag
     6241 ctgcatccgg actgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     6301 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     6361 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     6421 gtgtggaaaa tctctagcag catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     6481 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     6541 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     6601 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     6661 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     6721 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6781 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6841 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6901 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     6961 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     7021 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     7081 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     7141 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     7201 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     7261 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     7321 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     7381 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     7441 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     7501 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     7561 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     7621 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     7681 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     7741 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     7801 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     7861 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     7921 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7981 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     8041 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     8101 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     8161 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     8221 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
     8281 ccctatggtg cactctcagt acaatctg
//
