LOCUS       sequence-445291-        7331 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Make lentivirus to knock down human FMR1 gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7331)
  AUTHORS   Guo Y, Shen M, Dong Q, Mendez-Albelo NM, Huang SX, Sirois CL, Le J, 
            Li M, Jarzembowski ED, Schoeller KA, Stockton ME, Horner VL, Sousa 
            AMM, Gao Y, Levine JE, Wang D, Chang Q, Zhao X
  TITLE     Elevated levels of FMRP-target MAP1B impair human and mouse neuronal
            development and mouse social behaviors via autophagy pathway.
  JOURNAL   Nat Commun. 2023 Jun 26;14(1):3801. doi: 10.1038/s41467-023-39337-0.
  PUBMED    37365192
REFERENCE   2  (bases 1 to 7331)
  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: "Nat 
            Commun."; date: "2023-06-26"; volume: "14(1):3801. doi"; pages: " 
            10.1038/s41467-023-39337-0"
COMMENT     Sequence Label: pLV-shFMR1-mCherry
FEATURES             Location/Qualifiers
     source          1..7331
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        14..262
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     14..34
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     185..204
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        364..762
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     regulatory      765..774
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             771..1481
                     /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(916..934)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1184..1203
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    1486..2074
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1539..1559)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1957..1968)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2148..2381
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2453..2574
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2490..2509)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2544..2563
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2614..2749
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2782..2798)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2782..2798)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2795..2817)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2806..2822
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2830..2836,2837..2854,2855..2860))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2875..2896
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3013..3030)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3184..3772)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(3943..4734,4735..4803))
                     /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     4566..4585
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4804..4908)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4976..4994
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5032..5054)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5154..5173
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5367..5389
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5381..5398
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5382..5398
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5413..5639
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5640..5820
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5867..5992
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6485..6718
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6903..6947
                     /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    7214..7330
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 tcgatactag gaagagggcc tatttcccat gattccttca tatttgcata tacgatacaa
       61 ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa
      121 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
      181 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg gtttatatat
      241 cttgtggaaa ggacgaggat ccggcatgtg atgcaactta catcaagagt gtaagttgca
      301 tcacatgctt ttttggaatt ctcgacctcg agcagggaca gcagagatcc agtttggact
      361 agtggcctcc gcgccgggtt ttggcgcctc ccgcgggcgc ccccctcctc acggcgagcg
      421 ctgccacgtc agacgaaggg cgcagcgagc gtcctgatcc ttccgcccgg acgctcagga
      481 cagcggcccg ctgctcataa gactcggcct tagaacccca gtatcagcag aaggacattt
      541 taggacggga cttgggtgac tctagggcac tggttttctt tccagagagc ggaacaggcg
      601 aggaaaagta gtcccttctc ggcgattctg cggagggatc tccgtggggc ggtgaacgcc
      661 gatgattata taaggacgcg ccgggtgtgg cacagctagt tccgtcgcag ccgggatttg
      721 ggtcgcggtt cttgtttgtg gatcgctgtg atcgtcactt ggcggccgcc atggtgagca
      781 agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg
      841 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
      901 agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg
      961 acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca
     1021 tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     1081 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca
     1141 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga
     1201 agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg
     1261 gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga
     1321 ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt
     1381 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg
     1441 gccgccactc caccggcggc atggacgagc tgtacaagta gcgacaatca acctctggat
     1501 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     1561 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     1621 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     1681 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     1741 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     1801 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     1861 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
     1921 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     1981 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     2041 acgagtcgga tctccctttg ggccgcctcc ccgcctggta cctttaagac caatgactta
     2101 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat
     2161 tcactcccaa cgaagataag atctgctttt tgcttgtact gggtctctct ggttagacca
     2221 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     2281 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     2341 atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct
     2401 tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt
     2461 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     2521 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     2581 ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     2641 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     2701 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct agacttttgc
     2761 agagaccaaa ttcgtaatca tgtcatagct gtttcctgtg tgaaattgtt atccgctcac
     2821 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     2881 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     2941 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3001 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3061 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3121 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3181 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3241 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3301 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3361 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3421 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     3481 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     3541 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3601 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     3661 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3721 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3781 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     3841 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     3901 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     3961 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4021 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4081 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4141 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     4201 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     4261 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     4321 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     4381 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     4441 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     4501 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     4561 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     4621 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     4681 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     4741 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     4801 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     4861 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     4921 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     4981 gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     5041 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     5101 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc
     5161 agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag
     5221 gagaaaatac cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg
     5281 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg
     5341 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtgc
     5401 caagctgacg cgtgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga
     5461 gttagcaaca tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa
     5521 ggtggtacga tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa
     5581 ccactgaatt gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg
     5641 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
     5701 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     5761 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     5821 gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
     5881 gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
     5941 aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa
     6001 gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
     6061 atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc
     6121 tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct
     6181 tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt
     6241 gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca
     6301 aaacaaaagt aagaccaccg cacagcaagc ggccactgat cttcagacct ggaggaggag
     6361 atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
     6421 taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg
     6481 gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt
     6541 caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca
     6601 atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
     6661 agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg
     6721 ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
     6781 ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag
     6841 aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag
     6901 aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta
     6961 acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
     7021 gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac
     7081 cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
     7141 aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac
     7201 ggtatcggtt aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa
     7261 tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attaaaaaat
     7321 tcaaaatttt a
//
