LOCUS       sequence-412518-        5808 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knockout of Map3K12 (DLK) and Map3K13 (LZK) using tandem sgRNAs, 
            expresses mCherry under the hSyn1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5808)
  AUTHORS   Duncan GJ, Ingram SD, Emberley K, Hill J, Cordano C, Abdelhak A, 
            McCane M, Jenks JE, Jabassini N, Ananth K, Ferrara SJ, Stedelin B, 
            Sivyer B, Aicher SA, Scanlan TS, Watkins TA, Mishra A, Nelson JW, 
            Green AJ, Emery B
  TITLE     Remyelination protects neurons from DLK-mediated neurodegeneration.
  JOURNAL   Nat Commun. 2024 Oct 23;15(1):9148. doi: 10.1038/s41467-024-53429-5.
  PUBMED    39443516
REFERENCE   2  (bases 1 to 5808)
  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: "2024-10-23"; volume: "15(1):9148. doi"; pages: " 
            10.1038/s41467-024-53429-5"
COMMENT     Sequence Label: AAV-U6-sgMap3K13(1)-U6-sgMap3K12(1)-hSyn1-mCherry
FEATURES             Location/Qualifiers
     source          1..5808
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(107..126)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      920..1508
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1409..1428
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1570..1699
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        1731..1979
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        2000..2075
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2166..2414
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        2435..2510
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2618..3065
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             3097..3807
                     /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(3242..3260)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3510..3529
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4124..4600
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4323..4342)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4640..4780
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4640..4769
                     /label=AAV2 ITR
     rep_origin      4855..5310
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4942..4961)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5152..5173
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5327..5346)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5446..5468
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5506..5524)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5592..5696
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5697..5765,5766..5808,1..749)
                     /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"
ORIGIN
        1 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
       61 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      121 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      181 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      241 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      301 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      361 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      421 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      481 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      541 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      601 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      661 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      721 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
      781 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
      841 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
      901 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
      961 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1021 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     1081 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1141 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1201 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1261 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1321 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1381 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1441 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1501 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1561 ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg
     1621 gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac
     1681 tccatcacta ggggttcctg cggccgcacg cgttcgagaa tggcgcatgt gagggcctat
     1741 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
     1801 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
     1861 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
     1921 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacacct
     1981 ctggggaaca gcaacactgg ttttagagct agaaatagca agttaaaata aggctagtcc
     2041 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag ctagaaatag
     2101 caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag acaaatggct
     2161 ctagagaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
     2221 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
     2281 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
     2341 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
     2401 aaggacgaaa cacctgtaga gagcacatca gcgggtttta gagctagaaa tagcaagtta
     2461 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttgttt
     2521 tagagctaga aatagcaagt taaaataagg ctagtccgtt tttagcgcgt gcgccaattc
     2581 tgcagacaaa tggggtacaa attgggccct gcgtatgagt gcaagtgggt tttaggacca
     2641 ggatgaggcg gggtgggggt gcctacctga cgaccgaccc cgacccactg gacaagcacc
     2701 caacccccat tccccaaatt gcgcatcccc tatcagagag ggggagggga aacaggatgc
     2761 ggcgaggcgc gtgcgcactg ccagcttcag caccgcggac agtgccttcg cccccgcctg
     2821 gcggcgcgcg ccaccgccgc ctcagcactg aaggcgcgct gacgtcactc gccggtcccc
     2881 cgcaaactcc ccttcccggc caccttggtc gcgtccgcgc cgccgccggc ccagccggac
     2941 cgcaccacgc gaggcgcgag ataggggggc acgggcgcga ccatctgcgc tgcggcgccg
     3001 gcgactcagc gctgcctcag tctgcggtgg gcagcggagg agtcgtgtcg tgcctgagag
     3061 cgcagtcgag aaggtaccgg atcctctaga gtcgacatgg tgagcaaggg cgaggaggat
     3121 aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac
     3181 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
     3241 gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct
     3301 cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg
     3361 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
     3421 gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag
     3481 ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg
     3541 gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag
     3601 aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc
     3661 aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc
     3721 cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc
     3781 ggcggcatgg acgagctgta caagtagtcc ggactcagat ctcgagagga ggaggaggag
     3841 acagacagca ggatgcccca cctcgacagc cccggcagct cccagccgag acgctccttc
     3901 ctctcaaggg tgatcagggc agcgctaccg ttgcagctgc ttctgctgct gctgctgctc
     3961 ctggcctgcc tgctacctgc ctctgaagat gactacagct gcacccaggc caacaacttt
     4021 gcccgatcct tctaccccat gctgcggtac accaacgggc cacctcccac ctaggaattc
     4081 gatatcaagc ttatcgatac cgagcgctgc tcgagagatc tacgggtggc atccctgtga
     4141 cccctcccca gtgcctctcc tggccctgga agttgccact ccagtgccca ccagccttgt
     4201 cctaataaaa ttaagttgca tcattttgtc tgactaggtg tccttctata atattatggg
     4261 gtggaggggg gtggtatgga gcaaggggca agttgggaag acaacctgta gggcctgcgg
     4321 ggtctattgg gaaccaagct ggagtgcagt ggcacaatct tggctcactg caatctccgc
     4381 ctcctgggtt caagcgattc tcctgcctca gcctcccgag ttgttgggat tccaggcatg
     4441 catgaccagg ctcagctaat ttttgttttt ttggtagaga cggggtttca ccatattggc
     4501 caggctggtc tccaactcct aatctcaggt gatctaccca ccttggcctc ccaaattgct
     4561 gggattacag gcgtgaacca ctgctccctt ccctgtcctt ctgattttgt aggtaaccac
     4621 gtgcggaccg agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc
     4681 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg
     4741 gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt
     4801 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
     4861 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     4921 ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     4981 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     5041 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
     5101 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     5161 tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga
     5221 ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     5281 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
     5341 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     5401 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     5461 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     5521 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     5581 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     5641 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     5701 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     5761 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagt
//
