LOCUS       sequence-342961-        3426 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To clone and express a gRNA in Anopheles.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3426)
  AUTHORS   Klug D, Arnold K, Mela-Lopez R, Marois E, Blandin SA
  TITLE     A toolbox of engineered mosquito lines to study salivary gland 
            biology and malaria transmission.
  JOURNAL   PLoS Pathog. 2022 Oct 12;18(10):e1010881. doi: 
            10.1371/journal.ppat.1010881. eCollection 2022 Oct.
  PUBMED    36223382
REFERENCE   2  (bases 1 to 3426)
  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: "PLoS 
            Pathog."; date: "2022-10-12"; volume: "18(10):e1010881. doi"; pages:
            " 10.1371/journal.ppat.1010881. eCollection 2022 Oct"
COMMENT     Sequence Label: pKSB-610 U6-sgRNA2 GGAA AGAG
FEATURES             Location/Qualifiers
     source          1..3426
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     674..690
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     674..690
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_RNA        complement(738..813)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1193..1209)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1194..1210)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(1238..1256)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1277..1293)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1277..1293)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1290..1312)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1301..1317
                     /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(1325..1331,1332..1349,1350..1355))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1370..1391
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1508..1525)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1679..2267)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1759..1778)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2438..3229,3230..3298))
                     /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     3061..3080
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3299..3403)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg agctccaccg
      661 cggtggcggc cgctctagaa ctagtggatc ccccggtctc gggaagatga aaataagaaa
      721 aacatttgac aaaaaaagca ccgactcggt gccacttttt caagttgata acggactagc
      781 cttattttaa cttgctattt ctagctctaa aacaggtctt ctcgaagacc caaggacgag
      841 gggaaaaaag gttgtatata tactttgcgc tttcaatcct tgctctagcg atgcataaag
      901 gatattcaag aaggattttt tgcctgagtg tgactgttat aaggttttga tcctgccgta
      961 actcacgcta atgtgatgtt tcataaactt ttcaacattt cttgtattgc ttcatctctt
     1021 ttttaatcaa aagttactat tcattgtaat acgttgaaat atcaaaaaag aataaacagt
     1081 tttcctatga ttaattcaaa aacagagttt tctagaataa ccaagagcca aaccgcttca
     1141 gtgtatatgt ggagagtgag accgggctgc aggaattcga tatcaagctt atcgataccg
     1201 tcgacctcga gggggggccc ggtaccagct tttgttccct ttagtgaggg ttaatttcga
     1261 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     1321 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     1381 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     1441 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     1501 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     1561 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     1621 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     1681 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     1741 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     1801 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     1861 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     1921 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     1981 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     2041 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     2101 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     2161 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     2221 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     2281 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     2341 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     2401 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     2461 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     2521 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     2581 atccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     2641 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     2701 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     2761 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     2821 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     2881 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     2941 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     3001 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     3061 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     3121 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     3181 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     3241 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     3301 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     3361 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     3421 tgccac
//
