LOCUS       sequence-333384-        4606 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of gRNA in Culex quinquefasciatus or other mosquitoes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4606)
  AUTHORS   Feng X, Lopez Del Amo V, Mameli E, Lee M, Bishop AL, Perrimon N, 
            Gantz VM
  TITLE     Optimized CRISPR tools and site-directed transgenesis towards gene 
            drive development in Culex quinquefasciatus mosquitoes.
  JOURNAL   Nat Commun. 2021 May 20;12(1):2960. doi: 10.1038/s41467-021-23239-0.
  PUBMED    34017003
REFERENCE   2  (bases 1 to 4606)
  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: "Nat 
            Commun."; date: "2021-05-20"; volume: "12(1):2960. doi"; pages: " 
            10.1038/s41467-021-23239-0"
COMMENT     Sequence Label: pVMG0149_Cq-U6-6_2xBbsI-gRNA
FEATURES             Location/Qualifiers
     source          1..4606
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(76..95)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     286..307
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             761..1555
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(815..834)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1425..1444
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(1573..1641,1642..2433)
                     /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     complement(1791..1810)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2604..3192
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3093..3112
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3346..3363
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3480..3501
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3516..3521,3522..3539,3540..3546)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3554..3570
                     /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)."
     primer_bind     3559..3581
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3578..3594
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3578..3594
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_RNA        4259..4334
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(4412..4431)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4413..4431)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4438..4455)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4438..4454)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4447..4469)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      join(4595..4606,1..417)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
       61 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
      121 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
      181 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
      241 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
      301 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
      361 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
      421 acaaaattca gggcgcaagg gctgctaaag gaagcggaac acgtagaaag ccagtccgca
      481 gaaacggtgc tgaccccgga tgaatgtcag ctactgggct atctggacaa gggaaaacgc
      541 aagcgcaaag agaaagcagg tagcttgcag tgggcttaca tggcgatagc tagactgggc
      601 ggttttatgg acagcaagcg aaccggaatt gccagctggg gcgccctctg gtaaggttgg
      661 gaagccctgc aaagtaaact ggatggcttt cttgccgcca aggatctgat ggcgcagggg
      721 atcaagatct gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
      781 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
      841 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
      901 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
      961 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     1021 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catcccacct
     1081 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     1141 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     1201 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     1261 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
     1321 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     1381 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     1441 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     1501 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgaattga
     1561 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     1621 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     1681 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     1741 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     1801 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     1861 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     1921 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     1981 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     2041 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     2101 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     2161 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     2221 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     2281 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     2341 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     2401 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     2461 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     2521 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     2581 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     2641 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2701 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     2761 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     2821 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     2881 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     2941 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     3001 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     3061 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3121 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3181 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3241 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     3301 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     3361 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     3421 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     3481 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     3541 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     3601 tacgccaagc ttggtaccga gctcggatcc actagtaacg gccgccagtg tgctggaatt
     3661 cgcccttcaa aatctacgaa gataataagg tctactatta catttaataa atattcttat
     3721 ttaatttgag ctgtgtaaaa ttagtgcgtt tatccgggga atgtttatat tttatccgag
     3781 gatttcccga aattgattaa aaaaatcaaa tctcttaaaa aattagaaac aacctgttaa
     3841 aaataagctt atagaaaaaa tgtaaagtgt tgctacaagg acaatcaatt aatcatcata
     3901 acgaaaactt ttaggatctt gaaaagcacg tcccgagatg ggaaagtgtg tttcatttcc
     3961 aggcgatcct tttcgcttca cgaaattttt gattgctacc tcgtttagag ccctaagaca
     4021 aaatacttct tcaaaaaaaa gcgaactcga acccaaactt tgtactacta tgctaaaatg
     4081 tctgcatgca tgggatgtcg cgcaaacgag tccaataaga aataaaactg ctcgaattca
     4141 acgtgacgct acccgcaatg ctctaccctt aactttatac atcgctagaa ccgcgttcgc
     4201 attcgctaac tatataagca ctttcccggc cctacaactt gggtcttcga gaagacctgt
     4261 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
     4321 caccgagtcg gtgctttttt tttagggcga attctgcaga tatccatcac actggcggcc
     4381 gctcgagcat gcatctagag ggcccaattc gccctatagt gagtcgtatt acaattcact
     4441 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     4501 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     4561 ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc cctgta
//
