LOCUS       sequence-258857-        3459 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes gRNA targeting Drosophila crk locus. Cuts 110bp upstream of 
            the transcription start site. gRNA cloned into BbsI site of 
            pU6-BbsI-chiRNA (Gratz et al. Genetics. 2013)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3459)
  AUTHORS   Spracklen AJ, Thornton-Kolbe EM, Bonner AN, Florea A, Compton PJ, 
            Fernandez-Gonzalez R, Peifer M
  TITLE     The Crk adapter protein is essential for Drosophila embryogenesis, 
            where it regulates multiple actin-dependent morphogenic events.
  JOURNAL   Mol Biol Cell. 2019 Jul 18:mbcE19050302. doi: 
            10.1091/mbc.E19-05-0302.
  PUBMED    31318326
REFERENCE   2  (bases 1 to 3459)
  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: "Mol Biol 
            Cell. 2019 Jul 18:mbcE19050302. doi: 10.1091/mbc.E19-05-0302."
COMMENT     Sequence Label: pU6-chiRNA_dCrkgRNA1
FEATURES             Location/Qualifiers
     source          1..3459
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      7..435
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(94..113)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     304..325
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     669..685
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     670..686
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        698..1097
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
     misc_RNA        1117..1192
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1221..1237)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1221..1237)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(1274..1292)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1313..1329)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1313..1329)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1326..1348)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1337..1353
                     /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)."
     promoter        complement(join(1361..1367,1368..1385,1386..1391))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1406..1427
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1544..1561)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1715..2303)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1795..1814)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2474..3265,3266..3334))
                     /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     3097..3116
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3335..3439)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
       61 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
      121 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
      181 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
      241 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
      301 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
      361 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
      421 aatttaacgc gaattttaac aaaatattct acagggcgcg tcccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tgggtaccgg
      661 gccccccctc gaggtcgacg gtatcgataa gcttgatgtt cgacttgcag cctgaaatac
      721 ggcacgagta ggaaaagccg agtcaaatgc cgaatgcaga gtctcattac agcacaatca
      781 actcaagaaa aactcgacac ttttttacca tttgcactta aatccttttt tattcgttat
      841 gtatactttt tttggtccct aaccaaaaca aaaccaaact ctcttagtcg tgcctctata
      901 tttaaaacta tcaatttatt atagtcaata aatcgaactg tgttttcaac aaacgaacaa
      961 taggacactt tgattctaaa ggaaattttg aaaatcttaa gcagagggtt cttaagacca
     1021 tttgccaatt cttataattc tcaactgctc tttcctgatg ttgatcattt atataggtat
     1081 gttttcctca atacttcgct gacagtacgt cctaaagttt tagagctaga aatagcaagt
     1141 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttat
     1201 cgaattcctg cagcccgggg gatccactag ttctagagcg gccgccaccg cggtggagct
     1261 ccagcttttg ttccctttag tgagggttaa tttcgagctt ggcgtaatca tggtcatagc
     1321 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     1381 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     1441 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     1501 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     1561 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     1621 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     1681 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     1741 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     1801 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     1861 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     1921 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     1981 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     2041 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     2101 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     2161 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     2221 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     2281 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     2341 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     2401 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     2461 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     2521 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     2581 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     2641 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     2701 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     2761 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     2821 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     2881 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     2941 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     3001 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     3061 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     3121 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     3181 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     3241 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     3301 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     3361 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     3421 aacaaatagg ggttccgcgc acatttcccc tgaatggcg
//
