LOCUS       sequence-258784-        3460 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes gRNA targeting Drosophila crk locus. Cuts 99bp beyond the 3'
            UTR. 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 3460)
  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 3460)
  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_dCrkgRNA2
FEATURES             Location/Qualifiers
     source          1..3460
                     /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        1118..1193
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1222..1238)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1222..1238)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(1275..1293)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1314..1330)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1314..1330)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1327..1349)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1338..1354
                     /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(1362..1368,1369..1386,1387..1392))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1407..1428
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1545..1562)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1716..2304)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1796..1815)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2475..3266,3267..3335))
                     /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     3098..3117
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3336..3440)
                     /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 atacttcgaa ttgtgtaact aaacggtgtt ttagagctag aaatagcaag
     1141 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgctttttta
     1201 tcgaattcct gcagcccggg ggatccacta gttctagagc ggccgccacc gcggtggagc
     1261 tccagctttt gttcccttta gtgagggtta atttcgagct tggcgtaatc atggtcatag
     1321 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     1381 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     1441 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     1501 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     1561 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     1621 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     1681 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     1741 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     1801 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     1861 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     1921 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     1981 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     2041 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     2101 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     2161 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     2221 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     2281 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     2341 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     2401 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     2461 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     2521 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     2581 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     2641 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     2701 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     2761 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     2821 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     2881 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     2941 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     3001 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     3061 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     3121 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     3181 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     3241 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     3301 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     3361 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3421 aaacaaatag gggttccgcg cacatttccc ctgaatggcg
//
