LOCUS       sequence-419849-        8246 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pDEL111; Gateway Destination vector for cell-type specific CRISPR 
            encoding nrg1 type I 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_T1_gRNA
FEATURES             Location/Qualifiers
     source          1..8246
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        6..110
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(111..179,180..971)
                     /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(329..348)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1142..1730
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1631..1650
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1884..1901
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2018..2039
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2054..2059,2060..2077,2078..2084)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2092..2108
                     /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     2097..2119
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2116..2132
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2151..2171
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2153..2171
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2208..2224
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2208..2224
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    join(2258..2269,2270..2457)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     CDS             join(3679..3681,3682..3684,3685..4398)
                     /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(3679..3681,3682..3684,3685..4395)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3724..3745)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3985..4004)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4332..4353
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4700..4834
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_RNA        5219..5294
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     5307..5323
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5331..5454
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(5479..5484,5485..5502,5503..5509)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             5563..6243
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(5629..5648)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             6563..6868
                     /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     6747..6766
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(6909..7032)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(7052..7068)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    join(7441..7603,7604..7615)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     8068..8089
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
       61 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
      121 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
      181 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
      241 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
      301 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
      361 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
      421 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
      481 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
      541 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
      601 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
      661 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
      721 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
      781 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
      841 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
      901 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
      961 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     1021 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatgc
     1081 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     1141 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     1201 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     1261 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     1321 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     1381 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     1441 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     1501 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     1561 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     1621 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     1681 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     1741 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     1801 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     1861 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     1921 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     1981 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
     2041 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     2101 gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc
     2161 ctcactaaag ggaacaaaag ctggagctcc accgcggtgg cggccgctct agaactagtg
     2221 gatctgctgg gcttgctgaa ggtagggggt caagaaccag aggtgtaaag tacttgagta
     2281 attttacttg attactgtac ttaagtatta tttttgggga tttttacttt acttgagtac
     2341 aattaaaaat caatactttt acttttactt aattacattt ttttagaaaa aaaagtactt
     2401 tttactcctt acaattttat ttacagtcaa aaagtactta ttttttggag atcacttcat
     2461 tctattttcc cttgctatta ccaaaccaat tgaattgcgc tgatgcccag tttaatttaa
     2521 atgttattta ttctgcctat gaaaatcgtt ttcacattat atgaaattgg tcagacatgt
     2581 tcattggtcc tttggaagtg acgtcatgtc acatctatta ccacaatgca cagcaccttg
     2641 acctggaaat tagggaaatt ataacagtca atcagtggaa gaaaatggag gaagtatgtg
     2701 attcatcagc agctgcgagc agcacagtcc aaaatcagcc acaggatcaa gagcacccgt
     2761 ggccgtatct tcgcagatct aaagcttaaa tcagttgtgt taaataagag acattcaaaa
     2821 taaatgtaaa tgagctctcc aaatcagcag acttaacatt ctttaaaatg attgattcaa
     2881 tagtgataaa aatcaggcat agccagttgt aactttagat aaattacaga aaatgtcaaa
     2941 tacagagaac cgattctttt ttatgataca tccaagcaca catttaacac aatccaggca
     3001 aaccccgaat ttcacagtca caagcactgt ttgtacaaga gctttgccta aggacacaca
     3061 gtctctataa gtccaggtcg ttggtttcac tcttatttta aacatgtgac atttttcctg
     3121 ccatcctgtc ttaggctgct gtttgcttca ttccatgtca cattaaattc ctcagtagca
     3181 ccttttacac acacagccaa tcttttccag aaaattcaat tgctttgaag agataatgtg
     3241 tgaacaaatc catttagaaa aggaaaatta agaatttgta aaatcatctg taaattgttg
     3301 gcattcttct gtatatgaac atcacatcat ttacaggtaa aggtctggtc attaattata
     3361 tgacaattta ctggtattat tttgtgaaag gggctatttt caatgcgttc atccatcctt
     3421 ttcatccctc aaatctctca ttcacgtccc cctccccatc tgcacacttt atctcatttc
     3481 caccctgctg gaatctgagc acttgtgcag ttatcagggc tcctgtattt aggaggctct
     3541 gggtgtccat gtaggggacg aacagaaaca ctgcagacct ttatagaaga acaattgata
     3601 agagtcctca tacataaaga ctccattagt aagccagtga cccaggagcc cagaccaaca
     3661 gcaaagcaga cagtgaccat ggtgagcaag ggcgaagagc tgttcaccgg ggtggtgccc
     3721 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     3781 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     3841 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     3901 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     3961 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     4021 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     4081 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     4141 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     4201 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     4261 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     4321 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     4381 gacgagctgt acaagtaaag cggcccctct cgagcctcta gaactatagt gagtcgtatt
     4441 acgtagatcc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     4501 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     4561 taagctgcaa taaacaagag atctgatcta gaggatcata atcagccata ccacatttgt
     4621 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     4681 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     4741 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4801 caaactcatc aatgtatctt atcatgtctg gatcatcatc gatgcgtctt ttgttctggt
     4861 catcaaggag gggggaatgt tctgcgcatg cctgtggggg agggagaagg acacgtcact
     4921 gaaaacgtcc ctgcatcaca ccgagacacc caatcactca agccgagacc agataatttt
     4981 gcatatgctt taaagtttga aaaataccac ggtaaaccct cacacaaact ctggatttga
     5041 gatctttcag gttttatcag tttgcaggtt tatgtcacca tgatataggg tcagacttga
     5101 tctaagggag ctgaataagt ggtttagtca ctcaccacct cccaaaaaca tacccagaag
     5161 tccctggtat atatagctct ccctccagct cttggttcgc aggcaaagaa gggaaaaggt
     5221 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
     5281 caccgagtcg gtgctttttt ggtaccgtaa aacgacggcc agtgaattat caactatgta
     5341 taataaagtt gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt
     5401 gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac tatggcggcc
     5461 gcattaggca ccccaggctt tacactttat gcttccggct cgtataatgt gtggattttg
     5521 agttaggatc cgtcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact
     5581 ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag
     5641 tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag
     5701 accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg
     5761 atgaatgctc atccggaatt ccgtatggca atgaaagacg gtgagctggt gatatgggat
     5821 agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg
     5881 agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt
     5941 tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca
     6001 gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc
     6061 ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg
     6121 ctggcgattc aggttcatca tgccgtttgt gatggcttcc atgtcggcag aatgcttaat
     6181 gaattacaac agtactgcga tgagtggcag gcggggcgta atctagagga tccggcttac
     6241 taaaagccag ataacagtat gcgtatttgc gcgctgattt ttgcggtata agaatatata
     6301 ctgatatgta tacccgaagt atgtcaaaaa gaggtatgct atgaagcagc gtattacagt
     6361 gacagttgac agcgacagct atcagttgct caaggcatat atgatgtcaa tatctccggt
     6421 tcggtaagca caaccatgca gaatgaagcc cgtcgtctgc gtgccgaacg ctggaaagcg
     6481 gaaaatcagg aagggatggc tgaggtcgcc cggtttattg aaatgaacgg ctcctttgcc
     6541 gacgagaaca ggggctggtg aaatgcagtt taaggtttac acctataaaa gagagagccg
     6601 ttatcgtctg tttgtggatg tacagagtga tattattgac acgcccgggc gacggatggt
     6661 gatccccctg gccagtgcac gtctgctgtc agataaagtc ccccgtgaac tttacccggt
     6721 ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc gatatggcca gtgtgccggt
     6781 ctccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc
     6841 cattaacctg atgttctggg gaatataaat gtcaggctcc cttatacaca gccagtctgc
     6901 aggtcgacca tagtgactgg atatgttgtg ttttacagta ttatgtagtc tgttttttat
     6961 gcaaaatcta atttaatata ttgatattta tatcatttta cgtttctcgt tcaacttttc
     7021 tatacaaagt tgatagcttg gcgtaatcat ggtcatagct gtttcctgct cgagggccca
     7081 tctggcctgt gtttcagaca ccagggagtc tctgctcacg tttcctgcta tttgcagcct
     7141 ctctatcaag actaatacac ctcttcccgc atcggctgcc tgtgagaggc ttttcagcac
     7201 tgcaggattg cttttcagcc ccaaaagagc taggcttgac actaacaatt ttgagaatca
     7261 gcttctactg aagttaaatc tgaggtttta caactttgag tagcgtgtac tggcattaga
     7321 ttgtctgtct tatagtttga taattaaata caaacagttc taaagcagga taaaaccttg
     7381 tatgcatttc atttaatgtt ttttgagatt aaaagcttaa acaagaatct ctagttttct
     7441 ttcttgcttt tacttttact tccttaatac tcaagtacaa ttttaatgga gtactttttt
     7501 acttttactc aagtaagatt ctagccagat acttttactt ttaattgagt aaaattttcc
     7561 ctaagtactt gtactttcac ttgagtaaaa tttttgagta ctttttacac ctctgtcaag
     7621 aactcctgga caaacctctg acctgtgtgg aacagagtgg atatgggtgt ctgaacagat
     7681 attcacgtct tttgcagatc agagggcatt tctggtgttg tgcaatgacc tgggtccaac
     7741 aggtaggcgt actcgtatcc aagagcattg ttggacagga ggtagtctcc attcctataa
     7801 agaggaagga atggcaccat gtagtagccg tcattgtggc caattggtgc attggtacgt
     7861 gggtagatgg accggggagg ctgatgagtt cttagccagc ctttccccgt caagctctaa
     7921 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     7981 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     8041 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     8101 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     8161 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     8221 caatttaggt ggcacttttc ggggaa
//
