LOCUS       sequence-412380-        5808 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knockout of Map3K12 (DLK) 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-sgMap3K12(1)-U6-sgMap3K12(2)-hSyn1-mCherry
FEATURES             Location/Qualifiers
     source          1..5808
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(98..117)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      911..1499
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1400..1419
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1561..1690
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        1722..1970
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1991..2066
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2157..2405
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        2426..2501
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2609..3056
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             3088..3798
                     /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(3233..3251)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3501..3520
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4115..4591
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4314..4333)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4631..4771
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4631..4760
                     /label=AAV2 ITR
     rep_origin      4846..5301
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4933..4952)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5143..5164
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5318..5337)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5437..5459
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5497..5515)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5583..5687
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5688..5756,5757..5808,1..740)
                     /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 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
       61 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
      121 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
      181 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
      241 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
      301 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
      361 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
      421 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
      481 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
      541 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
      601 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
      661 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
      721 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
      781 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
      841 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
      901 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
      961 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     1021 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     1081 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     1141 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     1201 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     1261 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     1321 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     1381 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     1441 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     1501 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     1561 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
     1621 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
     1681 aggggttcct gcggccgcac gcgttcgaga atggcgcatg tgagggccta tttcccatga
     1741 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga
     1801 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
     1861 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
     1921 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc tgtagagagc
     1981 acatcagcgg gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
     2041 ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga gctagaaata gcaagttaaa
     2101 ataaggctag tccgttttta gcgcgtgcgc caattctgca gacaaatggc tctagagagg
     2161 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     2221 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     2281 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     2341 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
     2401 acaccagggt gttcgggttt catgggtttt agagctagaa atagcaagtt aaaataaggc
     2461 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt ttagagctag
     2521 aaatagcaag ttaaaataag gctagtccgt ttttagcgcg tgcgccaatt ctgcagacaa
     2581 atggggtaca aattgggccc tgcgtatgag tgcaagtggg ttttaggacc aggatgaggc
     2641 ggggtggggg tgcctacctg acgaccgacc ccgacccact ggacaagcac ccaaccccca
     2701 ttccccaaat tgcgcatccc ctatcagaga gggggagggg aaacaggatg cggcgaggcg
     2761 cgtgcgcact gccagcttca gcaccgcgga cagtgccttc gcccccgcct ggcggcgcgc
     2821 gccaccgccg cctcagcact gaaggcgcgc tgacgtcact cgccggtccc ccgcaaactc
     2881 cccttcccgg ccaccttggt cgcgtccgcg ccgccgccgg cccagccgga ccgcaccacg
     2941 cgaggcgcga gatagggggg cacgggcgcg accatctgcg ctgcggcgcc ggcgactcag
     3001 cgctgcctca gtctgcggtg ggcagcggag gagtcgtgtc gtgcctgaga gcgcagtcga
     3061 gaaggtaccg gatcctctag agtcgacatg gtgagcaagg gcgaggagga taacatggcc
     3121 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag
     3181 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
     3241 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg
     3301 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc
     3361 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     3421 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     3481 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc
     3541 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag
     3601 ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc
     3661 gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag
     3721 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     3781 gacgagctgt acaagtagtc cggactcaga tctcgagagg aggaggagga gacagacagc
     3841 aggatgcccc acctcgacag ccccggcagc tcccagccga gacgctcctt cctctcaagg
     3901 gtgatcaggg cagcgctacc gttgcagctg cttctgctgc tgctgctgct cctggcctgc
     3961 ctgctacctg cctctgaaga tgactacagc tgcacccagg ccaacaactt tgcccgatcc
     4021 ttctacccca tgctgcggta caccaacggg ccacctccca cctaggaatt cgatatcaag
     4081 cttatcgata ccgagcgctg ctcgagagat ctacgggtgg catccctgtg acccctcccc
     4141 agtgcctctc ctggccctgg aagttgccac tccagtgccc accagccttg tcctaataaa
     4201 attaagttgc atcattttgt ctgactaggt gtccttctat aatattatgg ggtggagggg
     4261 ggtggtatgg agcaaggggc aagttgggaa gacaacctgt agggcctgcg gggtctattg
     4321 ggaaccaagc tggagtgcag tggcacaatc ttggctcact gcaatctccg cctcctgggt
     4381 tcaagcgatt ctcctgcctc agcctcccga gttgttggga ttccaggcat gcatgaccag
     4441 gctcagctaa tttttgtttt tttggtagag acggggtttc accatattgg ccaggctggt
     4501 ctccaactcc taatctcagg tgatctaccc accttggcct cccaaattgc tgggattaca
     4561 ggcgtgaacc actgctccct tccctgtcct tctgattttg taggtaacca cgtgcggacc
     4621 gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     4681 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     4741 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
     4801 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     4861 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     4921 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     4981 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     5041 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     5101 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     5161 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
     5221 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     5281 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     5341 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     5401 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     5461 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     5521 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     5581 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5641 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     5701 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5761 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcac
//
