LOCUS       sequence-486416-        3460 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  CRISPR gRNA targeting the C-terminal end of Breathless for 
            cleavage..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3460)
  AUTHORS   Ho EK, Kim-Yip RP, Simpkins AG, Farahani PE, Oatman HR, Posfai E, 
            Shvartsman SY, Toettcher JE
  TITLE     A live-cell biosensor of in vivo receptor tyrosine kinase activity 
            reveals feedback regulation of a developmental gradient.
  JOURNAL   Cell Rep. 2025 Jul 22;44(7):115930. doi: 
            10.1016/j.celrep.2025.115930. Epub 2025 Jun 27.
  PUBMED    40581929
REFERENCE   2  (bases 1 to 3460)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Rep.";
            date: "2025-07-22"; volume: "44(7):115930. doi"; pages: " 
            10.1016/j.celrep.2025.115930. Epub 2025 Jun 27"
COMMENT     Sequence Label: pU6-BbsI-Btl
FEATURES             Location/Qualifiers
     source          1..3460
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     62..84
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     76..93
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     77..93
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     100..119
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        100..118
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     143..159
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     144..160
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        172..571
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
     misc_RNA        592..667
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(696..712)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(696..712)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(749..767)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(788..804)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(788..804)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(801..823)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    812..828
                     /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(836..842,843..860,861..866))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    881..902
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1019..1036)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1190..1778)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1270..1289)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1949..2740,2741..2809))
                     /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     2572..2591
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2810..2914)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2940..3395)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3077..3098)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3289..3308
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt
       61 tcccagtcac gacgttgtaa aacgacggcc agtgaattgt aatacgactc actatagggc
      121 gaattgggta ccgggccccc cctcgaggtc gacggtatcg ataagcttga tgttcgactt
      181 gcagcctgaa atacggcacg agtaggaaaa gccgagtcaa atgccgaatg cagagtctca
      241 ttacagcaca atcaactcaa gaaaaactcg acactttttt accatttgca cttaaatcct
      301 tttttattcg ttatgtatac tttttttggt ccctaaccaa aacaaaacca aactctctta
      361 gtcgtgcctc tatatttaaa actatcaatt tattatagtc aataaatcga actgtgtttt
      421 caacaaacga acaataggac actttgattc taaaggaaat tttgaaaatc ttaagcagag
      481 ggttcttaag accatttgcc aattcttata attctcaact gctctttcct gatgttgatc
      541 atttatatag gtatgttttc ctcaatactt cggtgtactg atatctaagc ggttttagag
      601 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      661 tcggtgcttt tttatcgaat tcctgcagcc cgggggatcc actagttcta gagcggccgc
      721 caccgcggtg gagctccagc ttttgttccc tttagtgagg gttaatttcg agcttggcgt
      781 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
      841 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
      901 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
      961 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     1021 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     1081 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     1141 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     1201 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     1261 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     1321 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     1381 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     1441 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     1501 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     1561 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     1621 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     1681 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     1741 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     1801 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     1861 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     1921 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     1981 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     2041 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     2101 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     2161 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     2221 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     2281 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     2341 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     2401 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     2461 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     2521 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     2581 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     2641 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     2701 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     2761 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     2821 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     2881 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta
     2941 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     3001 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     3061 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     3121 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     3181 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     3241 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     3301 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     3361 acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa
     3421 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
//
