LOCUS       sequence-382863-        8308 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Lentiviral construct with mCherry and Puro cassettes to express 
            sgRNA-9 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 Apr 14, 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_No9
FEATURES             Location/Qualifiers
     source          1..8308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        163..542
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        544..742
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             760..940
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    987..1112
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1609..1842
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2027..2071
                     /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    2369..2486
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2739..2772)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2776..2796
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     enhancer        3090..3393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3394..3597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3631..4227
                     /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(3631..3650)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4127..4147
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4237..4290
                     /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      4297..4306
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4303..5013
                     /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(4448..4466)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4716..4735
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(5033..5066)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5122..5710
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5175..5195)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5593..5604)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5713..5729)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5714..5730)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6239..6419
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6481..7069)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6561..6580)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7240..8031,8032..8100))
                     /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     7863..7882
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8101..8205)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8280..8299)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
       61 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
      121 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
      181 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      241 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      301 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      361 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      421 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      481 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      541 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
      601 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      661 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
      721 ggtgggaggt ctatataagc agcgcgtttt gcctgtactg ggtctctctg gttagaccag
      781 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
      841 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
      901 tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact
      961 tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc
     1021 gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag
     1081 gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc
     1141 gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag
     1201 tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag
     1261 aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac
     1321 ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa
     1381 aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg
     1441 cacagcaagc ggccggccgc tgatcttcag acctggagga ggagatatga gggacaattg
     1501 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac
     1561 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt
     1621 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt
     1681 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat
     1741 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag
     1801 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc
     1861 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct
     1921 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac
     1981 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga
     2041 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct
     2101 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt
     2161 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac
     2221 ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga
     2281 gagagacaga gacagatcca ttcgattagt gaacggatcg gcactgcgtg cgccaattct
     2341 gcagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca
     2401 gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa
     2461 aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga gatccagttt
     2521 ggttagtacc gggcccgctc tagagatccg acgcgccatc tctaggcccg cgccggcccc
     2581 ctcgcacgga cttgtgggag aagctcggct actcccctgc cccggttaat ttgcatataa
     2641 tatttcctag taactataga ggcttaatgt gcgataaaag acagataatc tgttcttttt
     2701 aatactagct acattttaca tgataggctt ggatttctat aacttcgtat agcatacatt
     2761 atacgaagtt ataaacagca caaaaggaaa ctcaccctaa ctgtaaagta attgtgtgtt
     2821 ttgagactat aagtatccct tggagaacca ccttgttggc ctacttgttc ctgctagatg
     2881 tttaagagct atgctggaaa cagcatagca agtttaaata aggctagtcc gttatcaact
     2941 tgaaaaagtg gcaccgagtc ggtgcttttt ttctcgagta ctaggatcca ttaggcggcc
     3001 gcgtggataa ccgtattacc gccatgcatt agttattaat agtaatcaat tacggggtca
     3061 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     3121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     3181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     3241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     3301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
     3361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
     3421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     3481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     3541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt
     3601 ttagtgaacc gtcagatccg ctagcccacc atgaccgagt acaagcccac ggtgcgcctc
     3661 gccacccgcg acgacgtccc ccgggccgta cgcaccctcg ccgccgcgtt cgccgactac
     3721 cccgccacgc gccacaccgt cgacccggac cgccacatcg agcgggtcac cgagctgcaa
     3781 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     3841 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     3901 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     3961 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     4021 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     4081 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     4141 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     4201 tggtgcatga cccgcaagcc cggtgccgga tcgggagagg gcagaggaag tctgctaaca
     4261 tgcggtgacg tcgaggagaa tcctggccca ccggtcgcca ccatggtgag caagggcgag
     4321 gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc
     4381 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
     4441 cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg
     4501 tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac
     4561 tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
     4621 ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag
     4681 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg
     4741 ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc
     4801 aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac
     4861 aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc
     4921 acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac
     4981 tccaccggcg gcatggacga gctgtacaag taagaattcg tcgagggacc taataacttc
     5041 gtatagcata cattatacga agttatacat gtttaagggt tccggttcca ctaggtacaa
     5101 ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact
     5161 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     5221 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     5281 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     5341 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     5401 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     5461 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     5521 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     5581 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     5641 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     5701 gcctccccgc atcgataccg tcgacctcga tcgagaccta gaaaaacatg gagcaatcac
     5761 aagtagcaat acagcagcta ccaatgctga ttgtgcctgg ctagaagcac aagaggagga
     5821 ggaggtgggt tttccagtca cacctcaggt acctttaaga ccaatgactt acaaggcagc
     5881 tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca
     5941 acgaagacaa gatatccttg atctgtggat ctaccacaca caaggctact tccctgattg
     6001 gcagaactac acaccagggc cagggatcag atatccactg acctttggat ggtgctacaa
     6061 gctagtacca gttgagcaag agaaggtaga agaagccaat gaaggagaga acacccgctt
     6121 gttacaccct gtgagcctgc atgggatgga tgacccggag agagaagtat tagagtggag
     6181 gtttgacagc cgcctagcat ttcatcacat ggcccgagag ctgcatccgg actgtactgg
     6241 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
     6301 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
     6361 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     6421 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     6481 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     6541 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     6601 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6661 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6721 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6781 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6841 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     6901 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     6961 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     7021 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     7081 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     7141 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     7201 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     7261 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     7321 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     7381 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     7441 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     7501 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     7561 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     7621 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     7681 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     7741 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     7801 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     7861 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     7921 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     7981 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     8041 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     8101 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     8161 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     8221 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
     8281 acaatctgct ctgatgccgc atagttaa
//
