LOCUS       sequence-419847-        8246 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pDEL112; Gateway Destination vector for cell-type specific CRISPR 
            encoding nrg1 type II 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_T2_gRNA
FEATURES             Location/Qualifiers
     source          1..8246
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        4..108
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(109..177,178..969)
                     /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(327..346)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1140..1728
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1629..1648
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1882..1899
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2016..2037
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2052..2057,2058..2075,2076..2082)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2090..2106
                     /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     2095..2117
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2114..2130
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2149..2169
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2151..2169
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2206..2222
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2206..2222
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    join(2256..2267,2268..2455)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     CDS             join(3677..3679,3680..3682,3683..4396)
                     /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(3677..3679,3680..3682,3683..4393)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3722..3743)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3983..4002)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4330..4351
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4698..4832
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_RNA        5217..5292
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     5305..5321
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5329..5452
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(5477..5482,5483..5500,5501..5507)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             5561..6241
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(5627..5646)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             6561..6866
                     /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     6745..6764
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(6907..7030)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(7050..7066)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    join(7439..7601,7602..7613)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     8066..8087
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
       61 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
      121 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
      181 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
      241 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
      301 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
      361 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
      421 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
      481 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
      541 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
      601 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
      661 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
      721 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
      781 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
      841 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
      901 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
      961 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     1021 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatgcct
     1081 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     1141 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     1201 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     1261 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     1321 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     1381 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     1441 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     1501 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     1561 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     1621 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     1681 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     1741 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     1801 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     1861 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     1921 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     1981 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     2041 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     2101 taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct
     2161 cactaaaggg aacaaaagct ggagctccac cgcggtggcg gccgctctag aactagtgga
     2221 tctgctgggc ttgctgaagg tagggggtca agaaccagag gtgtaaagta cttgagtaat
     2281 tttacttgat tactgtactt aagtattatt tttggggatt tttactttac ttgagtacaa
     2341 ttaaaaatca atacttttac ttttacttaa ttacattttt ttagaaaaaa aagtactttt
     2401 tactccttac aattttattt acagtcaaaa agtacttatt ttttggagat cacttcattc
     2461 tattttccct tgctattacc aaaccaattg aattgcgctg atgcccagtt taatttaaat
     2521 gttatttatt ctgcctatga aaatcgtttt cacattatat gaaattggtc agacatgttc
     2581 attggtcctt tggaagtgac gtcatgtcac atctattacc acaatgcaca gcaccttgac
     2641 ctggaaatta gggaaattat aacagtcaat cagtggaaga aaatggagga agtatgtgat
     2701 tcatcagcag ctgcgagcag cacagtccaa aatcagccac aggatcaaga gcacccgtgg
     2761 ccgtatcttc gcagatctaa agcttaaatc agttgtgtta aataagagac attcaaaata
     2821 aatgtaaatg agctctccaa atcagcagac ttaacattct ttaaaatgat tgattcaata
     2881 gtgataaaaa tcaggcatag ccagttgtaa ctttagataa attacagaaa atgtcaaata
     2941 cagagaaccg attctttttt atgatacatc caagcacaca tttaacacaa tccaggcaaa
     3001 ccccgaattt cacagtcaca agcactgttt gtacaagagc tttgcctaag gacacacagt
     3061 ctctataagt ccaggtcgtt ggtttcactc ttattttaaa catgtgacat ttttcctgcc
     3121 atcctgtctt aggctgctgt ttgcttcatt ccatgtcaca ttaaattcct cagtagcacc
     3181 ttttacacac acagccaatc ttttccagaa aattcaattg ctttgaagag ataatgtgtg
     3241 aacaaatcca tttagaaaag gaaaattaag aatttgtaaa atcatctgta aattgttggc
     3301 attcttctgt atatgaacat cacatcattt acaggtaaag gtctggtcat taattatatg
     3361 acaatttact ggtattattt tgtgaaaggg gctattttca atgcgttcat ccatcctttt
     3421 catccctcaa atctctcatt cacgtccccc tccccatctg cacactttat ctcatttcca
     3481 ccctgctgga atctgagcac ttgtgcagtt atcagggctc ctgtatttag gaggctctgg
     3541 gtgtccatgt aggggacgaa cagaaacact gcagaccttt atagaagaac aattgataag
     3601 agtcctcata cataaagact ccattagtaa gccagtgacc caggagccca gaccaacagc
     3661 aaagcagaca gtgaccatgg tgagcaaggg cgaagagctg ttcaccgggg tggtgcccat
     3721 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     3781 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     3841 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     3901 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     3961 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     4021 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     4081 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     4141 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     4201 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     4261 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     4321 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     4381 cgagctgtac aagtaaagcg gcccctctcg agcctctaga actatagtga gtcgtattac
     4441 gtagatccag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag
     4501 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
     4561 agctgcaata aacaagagat ctgatctaga ggatcataat cagccatacc acatttgtag
     4621 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     4681 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     4741 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     4801 aactcatcaa tgtatcttat catgtctgga tcatcatcga tgcgtctttt gttctggtca
     4861 tcaaggaggg gggaatgttc tgcgcatgcc tgtgggggag ggagaaggac acgtcactga
     4921 aaacgtccct gcatcacacc gagacaccca atcactcaag ccgagaccag ataattttgc
     4981 atatgcttta aagtttgaaa aataccacgg taaaccctca cacaaactct ggatttgaga
     5041 tctttcaggt tttatcagtt tgcaggttta tgtcaccatg atatagggtc agacttgatc
     5101 taagggagct gaataagtgg tttagtcact caccacctcc caaaaacata cccagaagtc
     5161 cctggtatat atagctctcc ctccagctct tggttcggag ctcgcccgcc gctcgggttt
     5221 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
     5281 ccgagtcggt gcttttttgg taccgtaaaa cgacggccag tgaattatca actatgtata
     5341 ataaagttga acgagaaacg taaaatgata taaatatcaa tatattaaat tagattttgc
     5401 ataaaaaaca gactacataa tactgtaaaa cacaacatat ccagtcacta tggcggccgc
     5461 attaggcacc ccaggcttta cactttatgc ttccggctcg tataatgtgt ggattttgag
     5521 ttaggatccg tcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg
     5581 atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc
     5641 agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac
     5701 cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat
     5761 gaatgctcat ccggaattcc gtatggcaat gaaagacggt gagctggtga tatgggatag
     5821 tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag
     5881 tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta
     5941 cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc
     6001 caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt
     6061 cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct
     6121 ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga
     6181 attacaacag tactgcgatg agtggcaggc ggggcgtaat ctagaggatc cggcttacta
     6241 aaagccagat aacagtatgc gtatttgcgc gctgattttt gcggtataag aatatatact
     6301 gatatgtata cccgaagtat gtcaaaaaga ggtatgctat gaagcagcgt attacagtga
     6361 cagttgacag cgacagctat cagttgctca aggcatatat gatgtcaata tctccggttc
     6421 ggtaagcaca accatgcaga atgaagcccg tcgtctgcgt gccgaacgct ggaaagcgga
     6481 aaatcaggaa gggatggctg aggtcgcccg gtttattgaa atgaacggct cctttgccga
     6541 cgagaacagg ggctggtgaa atgcagttta aggtttacac ctataaaaga gagagccgtt
     6601 atcgtctgtt tgtggatgta cagagtgata ttattgacac gcccgggcga cggatggtga
     6661 tccccctggc cagtgcacgt ctgctgtcag ataaagtccc ccgtgaactt tacccggtgg
     6721 tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga tatggccagt gtgccggtct
     6781 ccgttatcgg ggaagaagtg gctgatctca gccaccgcga aaatgacatc aaaaacgcca
     6841 ttaacctgat gttctgggga atataaatgt caggctccct tatacacagc cagtctgcag
     6901 gtcgaccata gtgactggat atgttgtgtt ttacagtatt atgtagtctg ttttttatgc
     6961 aaaatctaat ttaatatatt gatatttata tcattttacg tttctcgttc aacttttcta
     7021 tacaaagttg atagcttggc gtaatcatgg tcatagctgt ttcctgctcg agggcccatc
     7081 tggcctgtgt ttcagacacc agggagtctc tgctcacgtt tcctgctatt tgcagcctct
     7141 ctatcaagac taatacacct cttcccgcat cggctgcctg tgagaggctt ttcagcactg
     7201 caggattgct tttcagcccc aaaagagcta ggcttgacac taacaatttt gagaatcagc
     7261 ttctactgaa gttaaatctg aggttttaca actttgagta gcgtgtactg gcattagatt
     7321 gtctgtctta tagtttgata attaaataca aacagttcta aagcaggata aaaccttgta
     7381 tgcatttcat ttaatgtttt ttgagattaa aagcttaaac aagaatctct agttttcttt
     7441 cttgctttta cttttacttc cttaatactc aagtacaatt ttaatggagt acttttttac
     7501 ttttactcaa gtaagattct agccagatac ttttactttt aattgagtaa aattttccct
     7561 aagtacttgt actttcactt gagtaaaatt tttgagtact ttttacacct ctgtcaagaa
     7621 ctcctggaca aacctctgac ctgtgtggaa cagagtggat atgggtgtct gaacagatat
     7681 tcacgtcttt tgcagatcag agggcatttc tggtgttgtg caatgacctg ggtccaacag
     7741 gtaggcgtac tcgtatccaa gagcattgtt ggacaggagg tagtctccat tcctataaag
     7801 aggaaggaat ggcaccatgt agtagccgtc attgtggcca attggtgcat tggtacgtgg
     7861 gtagatggac cggggaggct gatgagttct tagccagcct ttccccgtca agctctaaat
     7921 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     7981 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     8041 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     8101 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     8161 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     8221 atttaggtgg cacttttcgg ggaaat
//
