LOCUS       sequence-486418-        3461 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  CRISPR gRNA targeting the C-terminal end of Torso for cleavage..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3461)
  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 3461)
  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-Torso
FEATURES             Location/Qualifiers
     source          1..3461
                     /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        593..668
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(697..713)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(697..713)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(750..768)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(789..805)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(789..805)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(802..824)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    813..829
                     /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(837..843,844..861,862..867))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    882..903
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1020..1037)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1191..1779)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1271..1290)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1950..2741,2742..2810))
                     /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     2573..2592
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2811..2915)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2941..3396)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3078..3099)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3290..3309
                     /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 cgagcaatga ctattaattc aagttttaga
      601 gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
      661 gtcggtgctt ttttatcgaa ttcctgcagc ccgggggatc cactagttct agagcggccg
      721 ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaatttc gagcttggcg
      781 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
      841 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
      901 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
      961 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     1021 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     1081 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     1141 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     1201 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     1261 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     1321 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     1381 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     1441 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     1501 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     1561 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     1621 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     1681 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     1741 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     1801 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     1861 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     1921 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     1981 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     2041 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     2101 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     2161 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     2221 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     2281 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     2341 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     2401 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     2461 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     2521 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     2581 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     2641 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     2701 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     2761 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     2821 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     2881 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     2941 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     3001 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     3061 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     3121 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     3181 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     3241 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     3301 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     3361 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca
     3421 actgttggga agggcgatcg gtgcgggcct cttcgctatt a
//
