LOCUS       sequence-378847-        3651 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector for multiple sgRNA cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3651)
  AUTHORS   Duarte F, Ramosaj M, Hasanovic E, Regio S, Sipion M, Rey M, Deglon N
  TITLE     Semi-automated workflows to quantify AAV transduction in various 
            brain areas and predict gene editing outcome for neurological 
            disorders.
  JOURNAL   Mol Ther Methods Clin Dev. 2023 Mar 27;29:254-270. doi: 
            10.1016/j.omtm.2023.03.013. eCollection 2023 Jun 8.
  PUBMED    37090478
REFERENCE   2  (bases 1 to 3651)
  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: "Mol Ther 
            Methods Clin Dev."; date: "2023-03-27"; volume: "29:254-270. doi"; 
            pages: " 10.1016/j.omtm.2023.03.013. eCollection 2023 Jun 8"
COMMENT     Sequence Label: 
            pMK-AttL1-NotI/BamHI-U6-BsaI-tracrRNAopt-BamHI-U6-SapI-tracrRNAopt-N
            otI/XbaI-7sk-BsmBI-tracrRNA-XbaI-AttL2
FEATURES             Location/Qualifiers
     source          1..3651
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     286..308
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     300..317
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     301..317
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     328..347
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        328..346
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             351..362
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    378..477
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        506..754
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        886..1134
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1528..1603
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1651..1750)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1865..1882)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1975..2563)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2055..2074)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2737..3531)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(3532..3636)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
       61 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      121 tagaccgaga tagggttgag tggccgctac agggcgctcc cattcgccat tcaggctgcg
      181 caactgttgg gaagggcgtt tcggtgcggg cctcttcgct attacgccag ctggcgaaag
      241 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
      301 gtaaaacgac ggccagtgag cgcgacgtaa tacgactcac tatagggcga attgaaggaa
      361 ggccgtcaag gccgcatcaa ataatgattt tattttgact gatagtgacc tgttcgttgc
      421 aacaaattga tgagcaatgc ttttttataa tgccaacttt gtacaaaaaa gcaggcttca
      481 tcgatcaatg cggccgctgg gatccgaggg cctatttccc atgattcctt catatttgca
      541 tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat
      601 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
      661 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
      721 ggctttatat atcttgtgga aaggacgaaa caccggagac cacggcaggt ctcagtttca
      781 gagctatgct ggaaacagca tagcaagttg aaataaggct agtccgttat caacttgaaa
      841 aagtggcacc gagtcggtgc tttttttgga tccaagcttc catgggaggg cctatttccc
      901 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
      961 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     1021 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     1081 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggaaga
     1141 gcgagctctt ctgtttcaga gctatgctgg aaacagcata gcaagttgaa ataaggctag
     1201 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttcatag ccatgggcgg
     1261 ccgctctaga ctgcagtatt tagcatgccc cacccatctg caaggcattc tggatagtgt
     1321 caaaacagcc ggaaatcaag tccgtttatc tcaaacttta gcattttggg aataaatgat
     1381 atttgctatg ctggttaaat tagattttag ttaaatttcc tgctgaagct ctagtacgat
     1441 aagtaacttg acctaagtgt aaagttgaga tttccttcag gtttatatag cttgtgcgcc
     1501 gcctgggtac ctcggagacg cgtctccgtt ttagagctag aaatagcaag ttaaaataag
     1561 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ggtacctcta
     1621 gatggaagct tctcgagact agtaggcctg acccagcttt cttgtacaaa gttggcatta
     1681 taagaaagca ttgcttatca atttgttgca acgaacaggt cactatcagt caaaataaaa
     1741 tcattatttg ctgggcctca tgggccttcc tttcactgcc cgctttccag tcgggaaacc
     1801 tgtcgtgcca gctgcattaa catggtcata gctgtttcct tgcgtattgg gcgctctccg
     1861 cttcctcgct cactgactcg ctgcgctcgg tcgttcgggt aaagcctggg gtgcctaatg
     1921 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     1981 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     2041 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     2101 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     2161 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     2221 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     2281 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     2341 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     2401 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     2461 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     2521 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     2581 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     2641 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     2701 ctaaagtata tatgagtaaa cttggtctga cagttattag aaaaattcat ccagcagacg
     2761 ataaaacgca atacgctggc tatccggtgc cgcaatgcca tacagcacca gaaaacgatc
     2821 cgcccattcg ccgcccagtt cttccgcaat atcacgggtg gccagcgcaa tatcctgata
     2881 acgatccgcc acgcccagac ggccgcaatc aataaagccg ctaaaacggc cattttccac
     2941 cataatgttc ggcaggcacg catcaccatg ggtcaccacc agatcttcgc catccggcat
     3001 gctcgctttc agacgcgcaa acagctctgc cggtgccagg ccctgatgtt cttcatccag
     3061 atcatcctga tccaccaggc ccgcttccat acgggtacgc gcacgttcaa tacgatgttt
     3121 cgcctgatga tcaaacggac aggtcgccgg gtccagggta tgcagacgac gcatggcatc
     3181 cgccataatg ctcacttttt ctgccggcgc cagatggcta gacagcagat cctgacccgg
     3241 cacttcgccc agcagcagcc aatcacggcc cgcttcggtc accacatcca gcaccgccgc
     3301 acacggaaca ccggtggtgg ccagccagct cagacgcgcc gcttcatcct gcagctcgtt
     3361 cagcgcaccg ctcagatcgg ttttcacaaa cagcaccgga cgaccctgcg cgctcagacg
     3421 aaacaccgcc gcatcagagc agccaatggt ctgctgcgcc caatcatagc caaacagacg
     3481 ttccacccac gctgccgggc tacccgcatg caggccatcc tgttcaatca tactcttcct
     3541 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     3601 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa a
//
