LOCUS       sequence-207914-        8308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector to express sgRNA targeting 
            ""GGCGGCGGCGGCGGCGGCGG"" locus by SpCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8308)
  AUTHORS   Liu XS, Wu H, Krzisch M, Wu X, Graef J, Muffat J, Hnisz D, Li CH, 
            Yuan B, Xu C, Li Y, Vershkov D, Cacace A, Young RA, Jaenisch R
  TITLE     Rescue of Fragile X Syndrome Neurons by DNA Methylation Editing of 
            the FMR1 Gene.
  JOURNAL   Cell. 2018 Feb 8. pii: S0092-8674(18)30049-7. doi: 
            10.1016/j.cell.2018.01.012.
  PUBMED    29456084
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: "Cell. 2018 
            Feb 8. pii: S0092-8674(18)30049-7. doi: 10.1016/j.cell.2018.01.012."
COMMENT     Sequence Label: pgRNA-CGG
FEATURES             Location/Qualifiers
     source          1..8308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(211..244)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     248..268
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     enhancer        562..865
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        866..1069
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             1103..1699
                     /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(1103..1122)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1599..1619
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1709..1762
                     /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      1769..1778
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1775..2485
                     /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(1920..1938)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2188..2207
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(2505..2538)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2594..3182
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2647..2667)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3065..3076)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3185..3201)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3186..3202)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             3711..3891
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(3953..4541)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4033..4052)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4712..5503,5504..5572))
                     /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     5335..5354
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5573..5677)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5752..5771)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5943..6322
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6324..6522
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             6540..6720
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6767..6892
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7389..7622
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7807..7851
                     /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    8149..8266
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 ccgggcccgc tctagagatc cgacgcgcca tctctaggcc cgcgccggcc ccctcgcacg
       61 gacttgtggg agaagctcgg ctactcccct gccccggtta atttgcatat aatatttcct
      121 agtaactata gaggcttaat gtgcgataaa agacagataa tctgttcttt ttaatactag
      181 ctacatttta catgataggc ttggatttct ataacttcgt atagcataca ttatacgaag
      241 ttataaacag cacaaaagga aactcaccct aactgtaaag taattgtgtg ttttgagact
      301 ataagtatcc cttggagaac caccttgttg gggcggcggc ggcggcggcg ggtttaagag
      361 ctatgctgga aacagcatag caagtttaaa taaggctagt ccgttatcaa cttgaaaaag
      421 tggcaccgag tcggtgcttt ttttctcgag tactaggatc cattaggcgg ccgcgtggat
      481 aaccgtatta ccgccatgca ttagttatta atagtaatca attacggggt cattagttca
      541 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
      601 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
      661 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      721 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
      781 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
      841 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
      901 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
      961 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
     1021 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa
     1081 ccgtcagatc cgctagccca ccatgaccga gtacaagccc acggtgcgcc tcgccacccg
     1141 cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
     1201 gcgccacacc gtcgacccgg accgccacat cgagcgggtc accgagctgc aagaactctt
     1261 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt
     1321 ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc
     1381 gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct
     1441 ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga
     1501 ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg
     1561 cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg
     1621 gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat
     1681 gacccgcaag cccggtgccg gatcgggaga gggcagagga agtctgctaa catgcggtga
     1741 cgtcgaggag aatcctggcc caccggtcgc caccatggtg agcaagggcg aggaggataa
     1801 catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg
     1861 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc
     1921 caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca
     1981 gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa
     2041 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt
     2101 ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct
     2161 gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga
     2221 ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag
     2281 gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa
     2341 gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca
     2401 caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg
     2461 cggcatggac gagctgtaca agtaagaatt cgtcgaggga cctaataact tcgtatagca
     2521 tacattatac gaagttatac atgtttaagg gttccggttc cactaggtac aattcgatat
     2581 caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     2641 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     2701 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     2761 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     2821 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     2881 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     2941 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
     3001 tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     3061 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     3121 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     3181 gcatcgatac cgtcgacctc gatcgagacc tagaaaaaca tggagcaatc acaagtagca
     3241 atacagcagc taccaatgct gattgtgcct ggctagaagc acaagaggag gaggaggtgg
     3301 gttttccagt cacacctcag gtacctttaa gaccaatgac ttacaaggca gctgtagatc
     3361 ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac
     3421 aagatatcct tgatctgtgg atctaccaca cacaaggcta cttccctgat tggcagaact
     3481 acacaccagg gccagggatc agatatccac tgacctttgg atggtgctac aagctagtac
     3541 cagttgagca agagaaggta gaagaagcca atgaaggaga gaacacccgc ttgttacacc
     3601 ctgtgagcct gcatgggatg gatgacccgg agagagaagt attagagtgg aggtttgaca
     3661 gccgcctagc atttcatcac atggcccgag agctgcatcc ggactgtact gggtctctct
     3721 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     3781 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     3841 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agcatgtgag
     3901 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3961 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4021 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4081 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4141 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4201 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4261 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4321 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4381 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4441 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4501 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4561 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4621 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     4681 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     4741 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     4801 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     4861 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     4921 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     4981 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5041 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5101 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5161 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5221 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5281 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     5341 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5401 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5461 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     5521 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     5581 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     5641 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     5701 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
     5761 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
     5821 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
     5881 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
     5941 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
     6001 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     6061 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     6121 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     6181 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     6241 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     6301 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
     6361 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     6421 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     6481 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agcgcgtttt gcctgtactg
     6541 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
     6601 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     6661 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     6721 gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
     6781 gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
     6841 aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa
     6901 gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
     6961 atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc
     7021 tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct
     7081 tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt
     7141 gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca
     7201 aaacaaaagt aagaccaccg cacagcaagc ggccggccgc tgatcttcag acctggagga
     7261 ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa
     7321 ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca
     7381 gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca
     7441 gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag
     7501 aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc
     7561 atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc
     7621 ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct
     7681 agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac
     7741 agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag
     7801 caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg
     7861 tttaacataa caaattggct gtggtatata aaattattca taatgatagt aggaggcttg
     7921 gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat
     7981 tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga
     8041 atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatcg
     8101 gcactgcgtg cgccaattct gcagacaaat ggcagtattc atccacaatt ttaaaagaaa
     8161 aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat
     8221 acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag
     8281 ggacagcaga gatccagttt ggttagta
//
