LOCUS       sequence-342964-        3436 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 3436)
  TITLE     Marois lab kit for CRISPR and transgenesis in Anopheles and Aedes
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3436)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pKSB-631 U6-sgRNA3/4 AACA GCTT
FEATURES             Location/Qualifiers
     source          1..3436
                     /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"
     primer_bind     complement(1203..1219)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1204..1220)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(1248..1266)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1287..1303)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1287..1303)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1300..1322)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1311..1327
                     /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(1335..1341,1342..1359,1360..1365))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1380..1401
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1518..1535)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1689..2277)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1769..1788)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2448..3239,3240..3308))
                     /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     3071..3090
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3309..3413)
                     /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 gaacagatga aaataagaaa
      721 aacatttgac aaaaaaagca ccgactcggt gccacttttt caagttgata acggactagc
      781 cttatttcaa cttgctatgc tgtttccagc atagctctga aacaggtctt ctcgaagacc
      841 caaggacgag gggaaaaaag gttgtatata tactttgcgc tttcaatcct tgctctagcg
      901 atgcataaag gatattcaag aaggattttt tgcctgagtg tgactgttat aaggttttga
      961 tcctgccgta actcacgcta atgtgatgtt tcataaactt ttcaacattt cttgtattgc
     1021 ttcatctctt ttttaatcaa aagttactat tcattgtaat acgttgaaat atcaaaaaag
     1081 aataaacagt tttcctatga ttaattcaaa aacagagttt tctagaataa ccaagagcca
     1141 aaccgcttca gtgtatatgt gggcttagag accgggctgc aggaattcga tatcaagctt
     1201 atcgataccg tcgacctcga gggggggccc ggtaccagct tttgttccct ttagtgaggg
     1261 ttaatttcga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     1321 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     1381 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     1441 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     1501 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     1561 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     1621 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     1681 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     1741 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     1801 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     1861 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     1921 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     1981 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     2041 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     2101 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     2161 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     2221 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     2281 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     2341 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     2401 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     2461 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     2521 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     2581 ataccgcgag atccacgctc accggctcca gatttatcag caataaacca gccagccgga
     2641 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     2701 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     2761 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     2821 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     2881 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     2941 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     3001 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     3061 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     3121 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     3181 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     3241 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     3301 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     3361 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     3421 ccccgaaaag tgccac
//
