LOCUS       sequence-412246-        8246 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pDEL133; Gateway Destination vector for cell-type specific CRISPR 
            encoding erbb2 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_erbb2_gRNA171
FEATURES             Location/Qualifiers
     source          1..8246
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        32..136
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(137..205,206..997)
                     /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(355..374)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1168..1756
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1657..1676
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1910..1927
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2044..2065
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2080..2085,2086..2103,2104..2110)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2118..2134
                     /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     2123..2145
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2142..2158
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2177..2197
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2179..2197
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2234..2250
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2234..2250
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    join(2284..2295,2296..2483)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     CDS             join(3705..3707,3708..3710,3711..4424)
                     /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(3705..3707,3708..3710,3711..4421)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3750..3771)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4011..4030)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4358..4379
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4726..4860
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_RNA        5245..5320
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     5333..5349
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5357..5480
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(5505..5510,5511..5528,5529..5535)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             5589..6269
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(5655..5674)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             6589..6894
                     /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     6773..6792
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(6935..7058)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(7078..7094)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    join(7467..7629,7630..7641)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     8094..8115
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
       61 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
      121 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
      181 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
      241 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
      301 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
      361 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
      421 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
      481 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
      541 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
      601 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
      661 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
      721 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
      781 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
      841 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
      901 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
      961 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     1021 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     1081 ttttgataat ctcatgacca aaatgcctta acgtgagttt tcgttccact gagcgtcaga
     1141 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     1201 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     1261 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     1321 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     1381 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     1441 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     1501 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     1561 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     1621 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     1681 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     1741 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     1801 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     1861 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     1921 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     1981 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     2041 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     2101 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     2161 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctccaccg
     2221 cggtggcggc cgctctagaa ctagtggatc tgctgggctt gctgaaggta gggggtcaag
     2281 aaccagaggt gtaaagtact tgagtaattt tacttgatta ctgtacttaa gtattatttt
     2341 tggggatttt tactttactt gagtacaatt aaaaatcaat acttttactt ttacttaatt
     2401 acattttttt agaaaaaaaa gtacttttta ctccttacaa ttttatttac agtcaaaaag
     2461 tacttatttt ttggagatca cttcattcta ttttcccttg ctattaccaa accaattgaa
     2521 ttgcgctgat gcccagttta atttaaatgt tatttattct gcctatgaaa atcgttttca
     2581 cattatatga aattggtcag acatgttcat tggtcctttg gaagtgacgt catgtcacat
     2641 ctattaccac aatgcacagc accttgacct ggaaattagg gaaattataa cagtcaatca
     2701 gtggaagaaa atggaggaag tatgtgattc atcagcagct gcgagcagca cagtccaaaa
     2761 tcagccacag gatcaagagc acccgtggcc gtatcttcgc agatctaaag cttaaatcag
     2821 ttgtgttaaa taagagacat tcaaaataaa tgtaaatgag ctctccaaat cagcagactt
     2881 aacattcttt aaaatgattg attcaatagt gataaaaatc aggcatagcc agttgtaact
     2941 ttagataaat tacagaaaat gtcaaataca gagaaccgat tcttttttat gatacatcca
     3001 agcacacatt taacacaatc caggcaaacc ccgaatttca cagtcacaag cactgtttgt
     3061 acaagagctt tgcctaagga cacacagtct ctataagtcc aggtcgttgg tttcactctt
     3121 attttaaaca tgtgacattt ttcctgccat cctgtcttag gctgctgttt gcttcattcc
     3181 atgtcacatt aaattcctca gtagcacctt ttacacacac agccaatctt ttccagaaaa
     3241 ttcaattgct ttgaagagat aatgtgtgaa caaatccatt tagaaaagga aaattaagaa
     3301 tttgtaaaat catctgtaaa ttgttggcat tcttctgtat atgaacatca catcatttac
     3361 aggtaaaggt ctggtcatta attatatgac aatttactgg tattattttg tgaaaggggc
     3421 tattttcaat gcgttcatcc atccttttca tccctcaaat ctctcattca cgtccccctc
     3481 cccatctgca cactttatct catttccacc ctgctggaat ctgagcactt gtgcagttat
     3541 cagggctcct gtatttagga ggctctgggt gtccatgtag gggacgaaca gaaacactgc
     3601 agacctttat agaagaacaa ttgataagag tcctcataca taaagactcc attagtaagc
     3661 cagtgaccca ggagcccaga ccaacagcaa agcagacagt gaccatggtg agcaagggcg
     3721 aagagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     3781 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     3841 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
     3901 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     3961 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     4021 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     4081 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     4141 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
     4201 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     4261 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     4321 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     4381 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaagcggc ccctctcgag
     4441 cctctagaac tatagtgagt cgtattacgt agatccagac atgataagat acattgatga
     4501 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     4561 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagagatct gatctagagg
     4621 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
     4681 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
     4741 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4801 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
     4861 atcatcgatg cgtcttttgt tctggtcatc aaggaggggg gaatgttctg cgcatgcctg
     4921 tgggggaggg agaaggacac gtcactgaaa acgtccctgc atcacaccga gacacccaat
     4981 cactcaagcc gagaccagat aattttgcat atgctttaaa gtttgaaaaa taccacggta
     5041 aaccctcaca caaactctgg atttgagatc tttcaggttt tatcagtttg caggtttatg
     5101 tcaccatgat atagggtcag acttgatcta agggagctga ataagtggtt tagtcactca
     5161 ccacctccca aaaacatacc cagaagtccc tggtatatat agctctccct ccagctcttg
     5221 gttcccaacc ttctctccct gtgggtttta gagctagaaa tagcaagtta aaataaggct
     5281 agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttggta ccgtaaaacg
     5341 acggccagtg aattatcaac tatgtataat aaagttgaac gagaaacgta aaatgatata
     5401 aatatcaata tattaaatta gattttgcat aaaaaacaga ctacataata ctgtaaaaca
     5461 caacatatcc agtcactatg gcggccgcat taggcacccc aggctttaca ctttatgctt
     5521 ccggctcgta taatgtgtgg attttgagtt aggatccgtc gagattttca ggagctaagg
     5581 aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc
     5641 gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc
     5701 agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg
     5761 cctttattca cattcttgcc cgcctgatga atgctcatcc ggaattccgt atggcaatga
     5821 aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc
     5881 aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac
     5941 acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt
     6001 ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt
     6061 taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc aaatattata
     6121 cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg
     6181 gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag tggcaggcgg
     6241 ggcgtaatct agaggatccg gcttactaaa agccagataa cagtatgcgt atttgcgcgc
     6301 tgatttttgc ggtataagaa tatatactga tatgtatacc cgaagtatgt caaaaagagg
     6361 tatgctatga agcagcgtat tacagtgaca gttgacagcg acagctatca gttgctcaag
     6421 gcatatatga tgtcaatatc tccggttcgg taagcacaac catgcagaat gaagcccgtc
     6481 gtctgcgtgc cgaacgctgg aaagcggaaa atcaggaagg gatggctgag gtcgcccggt
     6541 ttattgaaat gaacggctcc tttgccgacg agaacagggg ctggtgaaat gcagtttaag
     6601 gtttacacct ataaaagaga gagccgttat cgtctgtttg tggatgtaca gagtgatatt
     6661 attgacacgc ccgggcgacg gatggtgatc cccctggcca gtgcacgtct gctgtcagat
     6721 aaagtccccc gtgaacttta cccggtggtg catatcgggg atgaaagctg gcgcatgatg
     6781 accaccgata tggccagtgt gccggtctcc gttatcgggg aagaagtggc tgatctcagc
     6841 caccgcgaaa atgacatcaa aaacgccatt aacctgatgt tctggggaat ataaatgtca
     6901 ggctccctta tacacagcca gtctgcaggt cgaccatagt gactggatat gttgtgtttt
     6961 acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga tatttatatc
     7021 attttacgtt tctcgttcaa cttttctata caaagttgat agcttggcgt aatcatggtc
     7081 atagctgttt cctgctcgag ggcccatctg gcctgtgttt cagacaccag ggagtctctg
     7141 ctcacgtttc ctgctatttg cagcctctct atcaagacta atacacctct tcccgcatcg
     7201 gctgcctgtg agaggctttt cagcactgca ggattgcttt tcagccccaa aagagctagg
     7261 cttgacacta acaattttga gaatcagctt ctactgaagt taaatctgag gttttacaac
     7321 tttgagtagc gtgtactggc attagattgt ctgtcttata gtttgataat taaatacaaa
     7381 cagttctaaa gcaggataaa accttgtatg catttcattt aatgtttttt gagattaaaa
     7441 gcttaaacaa gaatctctag ttttctttct tgcttttact tttacttcct taatactcaa
     7501 gtacaatttt aatggagtac ttttttactt ttactcaagt aagattctag ccagatactt
     7561 ttacttttaa ttgagtaaaa ttttccctaa gtacttgtac tttcacttga gtaaaatttt
     7621 tgagtacttt ttacacctct gtcaagaact cctggacaaa cctctgacct gtgtggaaca
     7681 gagtggatat gggtgtctga acagatattc acgtcttttg cagatcagag ggcatttctg
     7741 gtgttgtgca atgacctggg tccaacaggt aggcgtactc gtatccaaga gcattgttgg
     7801 acaggaggta gtctccattc ctataaagag gaaggaatgg caccatgtag tagccgtcat
     7861 tgtggccaat tggtgcattg gtacgtgggt agatggaccg gggaggctga tgagttctta
     7921 gccagccttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     7981 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     8041 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     8101 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     8161 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     8221 gaattttaac aaaatattaa cgctta
//
