LOCUS       sequence-412248-        8246 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pDEL113; Gateway Destination vector for cell-type specific CRISPR 
            encoding nrg1 type III sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8246)
  AUTHORS   Lysko DE, Talbot WS
  TITLE     Unmyelinated sensory neurons use Neuregulin signals to promote 
            myelination of interneurons in the CNS.
  JOURNAL   Cell Rep. 2022 Nov 15;41(7):111669. doi: 
            10.1016/j.celrep.2022.111669.
  PUBMED    36384112
REFERENCE   2  (bases 1 to 8246)
  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 Rep.";
            date: "2022-11-15"; volume: "41(7):111669. doi"; pages: " 
            10.1016/j.celrep.2022.111669"
COMMENT     Sequence Label: pDest_U6_A2_CG2_T3_gRNA
FEATURES             Location/Qualifiers
     source          1..8246
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..125
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(126..194,195..986)
                     /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     complement(344..363)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1157..1745
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1646..1665
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1899..1916
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2033..2054
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2069..2074,2075..2092,2093..2099)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2107..2123
                     /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)."
     primer_bind     2112..2134
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2131..2147
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2166..2186
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2168..2186
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2223..2239
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2223..2239
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    join(2273..2284,2285..2472)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     CDS             join(3694..3696,3697..3699,3700..4413)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3694..3696,3697..3699,3700..4410)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3739..3760)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4000..4019)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4347..4368
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4715..4849
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_RNA        5234..5309
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     5322..5338
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5346..5469
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(5494..5499,5500..5517,5518..5524)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             5578..6258
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(5644..5663)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             6578..6883
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     6762..6781
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(6924..7047)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(7067..7083)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    join(7456..7618,7619..7630)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     8083..8104
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
       61 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
      121 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
      181 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
      241 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
      301 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
      361 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
      421 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
      481 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
      541 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
      601 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
      661 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
      721 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
      781 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
      841 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
      901 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
      961 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     1021 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     1081 tcatgaccaa aatgccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1141 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1201 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1261 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     1321 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1381 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1441 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1501 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     1561 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     1621 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     1681 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     1741 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     1801 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     1861 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     1921 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     1981 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     2041 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     2101 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     2161 agcgcgcaat taaccctcac taaagggaac aaaagctgga gctccaccgc ggtggcggcc
     2221 gctctagaac tagtggatct gctgggcttg ctgaaggtag ggggtcaaga accagaggtg
     2281 taaagtactt gagtaatttt acttgattac tgtacttaag tattattttt ggggattttt
     2341 actttacttg agtacaatta aaaatcaata cttttacttt tacttaatta cattttttta
     2401 gaaaaaaaag tactttttac tccttacaat tttatttaca gtcaaaaagt acttattttt
     2461 tggagatcac ttcattctat tttcccttgc tattaccaaa ccaattgaat tgcgctgatg
     2521 cccagtttaa tttaaatgtt atttattctg cctatgaaaa tcgttttcac attatatgaa
     2581 attggtcaga catgttcatt ggtcctttgg aagtgacgtc atgtcacatc tattaccaca
     2641 atgcacagca ccttgacctg gaaattaggg aaattataac agtcaatcag tggaagaaaa
     2701 tggaggaagt atgtgattca tcagcagctg cgagcagcac agtccaaaat cagccacagg
     2761 atcaagagca cccgtggccg tatcttcgca gatctaaagc ttaaatcagt tgtgttaaat
     2821 aagagacatt caaaataaat gtaaatgagc tctccaaatc agcagactta acattcttta
     2881 aaatgattga ttcaatagtg ataaaaatca ggcatagcca gttgtaactt tagataaatt
     2941 acagaaaatg tcaaatacag agaaccgatt cttttttatg atacatccaa gcacacattt
     3001 aacacaatcc aggcaaaccc cgaatttcac agtcacaagc actgtttgta caagagcttt
     3061 gcctaaggac acacagtctc tataagtcca ggtcgttggt ttcactctta ttttaaacat
     3121 gtgacatttt tcctgccatc ctgtcttagg ctgctgtttg cttcattcca tgtcacatta
     3181 aattcctcag tagcaccttt tacacacaca gccaatcttt tccagaaaat tcaattgctt
     3241 tgaagagata atgtgtgaac aaatccattt agaaaaggaa aattaagaat ttgtaaaatc
     3301 atctgtaaat tgttggcatt cttctgtata tgaacatcac atcatttaca ggtaaaggtc
     3361 tggtcattaa ttatatgaca atttactggt attattttgt gaaaggggct attttcaatg
     3421 cgttcatcca tccttttcat ccctcaaatc tctcattcac gtccccctcc ccatctgcac
     3481 actttatctc atttccaccc tgctggaatc tgagcacttg tgcagttatc agggctcctg
     3541 tatttaggag gctctgggtg tccatgtagg ggacgaacag aaacactgca gacctttata
     3601 gaagaacaat tgataagagt cctcatacat aaagactcca ttagtaagcc agtgacccag
     3661 gagcccagac caacagcaaa gcagacagtg accatggtga gcaagggcga agagctgttc
     3721 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     3781 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     3841 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     3901 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     3961 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     4021 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     4081 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     4141 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     4201 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     4261 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     4321 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     4381 atcactctcg gcatggacga gctgtacaag taaagcggcc cctctcgagc ctctagaact
     4441 atagtgagtc gtattacgta gatccagaca tgataagata cattgatgag tttggacaaa
     4501 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     4561 tatttgtaac cattataagc tgcaataaac aagagatctg atctagagga tcataatcag
     4621 ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa
     4681 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg
     4741 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     4801 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatca tcatcgatgc
     4861 gtcttttgtt ctggtcatca aggagggggg aatgttctgc gcatgcctgt gggggaggga
     4921 gaaggacacg tcactgaaaa cgtccctgca tcacaccgag acacccaatc actcaagccg
     4981 agaccagata attttgcata tgctttaaag tttgaaaaat accacggtaa accctcacac
     5041 aaactctgga tttgagatct ttcaggtttt atcagtttgc aggtttatgt caccatgata
     5101 tagggtcaga cttgatctaa gggagctgaa taagtggttt agtcactcac cacctcccaa
     5161 aaacataccc agaagtccct ggtatatata gctctccctc cagctcttgg ttcgggacgg
     5221 tgacagctcc gaggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
     5281 aacttgaaaa agtggcaccg agtcggtgct tttttggtac cgtaaaacga cggccagtga
     5341 attatcaact atgtataata aagttgaacg agaaacgtaa aatgatataa atatcaatat
     5401 attaaattag attttgcata aaaaacagac tacataatac tgtaaaacac aacatatcca
     5461 gtcactatgg cggccgcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     5521 aatgtgtgga ttttgagtta ggatccgtcg agattttcag gagctaagga agctaaaatg
     5581 gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat
     5641 tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt
     5701 acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac
     5761 attcttgccc gcctgatgaa tgctcatccg gaattccgta tggcaatgaa agacggtgag
     5821 ctggtgatat gggatagtgt tcacccttgt tacaccgttt tccatgagca aactgaaacg
     5881 ttttcatcgc tctggagtga ataccacgac gatttccggc agtttctaca catatattcg
     5941 caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt tattgagaat
     6001 atgtttttcg tctcagccaa tccctgggtg agtttcacca gttttgattt aaacgtggcc
     6061 aatatggaca acttcttcgc ccccgttttc accatgggca aatattatac gcaaggcgac
     6121 aaggtgctga tgccgctggc gattcaggtt catcatgccg tttgtgatgg cttccatgtc
     6181 ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt ggcaggcggg gcgtaatcta
     6241 gaggatccgg cttactaaaa gccagataac agtatgcgta tttgcgcgct gatttttgcg
     6301 gtataagaat atatactgat atgtataccc gaagtatgtc aaaaagaggt atgctatgaa
     6361 gcagcgtatt acagtgacag ttgacagcga cagctatcag ttgctcaagg catatatgat
     6421 gtcaatatct ccggttcggt aagcacaacc atgcagaatg aagcccgtcg tctgcgtgcc
     6481 gaacgctgga aagcggaaaa tcaggaaggg atggctgagg tcgcccggtt tattgaaatg
     6541 aacggctcct ttgccgacga gaacaggggc tggtgaaatg cagtttaagg tttacaccta
     6601 taaaagagag agccgttatc gtctgtttgt ggatgtacag agtgatatta ttgacacgcc
     6661 cgggcgacgg atggtgatcc ccctggccag tgcacgtctg ctgtcagata aagtcccccg
     6721 tgaactttac ccggtggtgc atatcgggga tgaaagctgg cgcatgatga ccaccgatat
     6781 ggccagtgtg ccggtctccg ttatcgggga agaagtggct gatctcagcc accgcgaaaa
     6841 tgacatcaaa aacgccatta acctgatgtt ctggggaata taaatgtcag gctcccttat
     6901 acacagccag tctgcaggtc gaccatagtg actggatatg ttgtgtttta cagtattatg
     6961 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
     7021 ctcgttcaac ttttctatac aaagttgata gcttggcgta atcatggtca tagctgtttc
     7081 ctgctcgagg gcccatctgg cctgtgtttc agacaccagg gagtctctgc tcacgtttcc
     7141 tgctatttgc agcctctcta tcaagactaa tacacctctt cccgcatcgg ctgcctgtga
     7201 gaggcttttc agcactgcag gattgctttt cagccccaaa agagctaggc ttgacactaa
     7261 caattttgag aatcagcttc tactgaagtt aaatctgagg ttttacaact ttgagtagcg
     7321 tgtactggca ttagattgtc tgtcttatag tttgataatt aaatacaaac agttctaaag
     7381 caggataaaa ccttgtatgc atttcattta atgttttttg agattaaaag cttaaacaag
     7441 aatctctagt tttctttctt gcttttactt ttacttcctt aatactcaag tacaatttta
     7501 atggagtact tttttacttt tactcaagta agattctagc cagatacttt tacttttaat
     7561 tgagtaaaat tttccctaag tacttgtact ttcacttgag taaaattttt gagtactttt
     7621 tacacctctg tcaagaactc ctggacaaac ctctgacctg tgtggaacag agtggatatg
     7681 ggtgtctgaa cagatattca cgtcttttgc agatcagagg gcatttctgg tgttgtgcaa
     7741 tgacctgggt ccaacaggta ggcgtactcg tatccaagag cattgttgga caggaggtag
     7801 tctccattcc tataaagagg aaggaatggc accatgtagt agccgtcatt gtggccaatt
     7861 ggtgcattgg tacgtgggta gatggaccgg ggaggctgat gagttcttag ccagcctttc
     7921 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     7981 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     8041 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     8101 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     8161 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     8221 aaatattaac gcttacaatt taggtg
//
