LOCUS       sequence-333380-        4616 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 4616)
  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 4616)
  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: pVMG0303_Cq-U6-6_2xBbsI_gRNA-Loop
FEATURES             Location/Qualifiers
     source          1..4616
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      37..465
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(124..143)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     334..355
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             809..1603
                     /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(863..882)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1473..1492
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(1621..1689,1690..2481)
                     /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(1839..1858)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2652..3240
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3141..3160
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3394..3411
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3528..3549
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3564..3569,3570..3587,3588..3594)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3602..3618
                     /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     3607..3629
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3626..3642
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3626..3642
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4470..4489)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4471..4489)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4496..4513)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4496..4512)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4505..4527)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ccttcccaac agttgcgcag cctgaatggc gaatggacgc gccctgtagc ggcgcattaa
       61 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
      121 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
      181 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
      241 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
      301 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
      361 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
      421 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaattcagg
      481 gcgcaagggc tgctaaagga agcggaacac gtagaaagcc agtccgcaga aacggtgctg
      541 accccggatg aatgtcagct actgggctat ctggacaagg gaaaacgcaa gcgcaaagag
      601 aaagcaggta gcttgcagtg ggcttacatg gcgatagcta gactgggcgg ttttatggac
      661 agcaagcgaa ccggaattgc cagctggggc gccctctggt aaggttggga agccctgcaa
      721 agtaaactgg atggctttct tgccgccaag gatctgatgg cgcaggggat caagatctga
      781 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
      841 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
      901 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
      961 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     1021 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     1081 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tcccaccttg ctcctgccga
     1141 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     1201 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     1261 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     1321 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     1381 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     1441 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     1501 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     1561 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgaattgaaa aaggaagagt
     1621 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     1681 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     1741 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     1801 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     1861 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     1921 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     1981 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     2041 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     2101 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     2161 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     2221 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     2281 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     2341 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     2401 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     2461 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     2521 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     2581 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     2641 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     2701 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     2761 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     2821 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     2881 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     2941 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     3001 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     3061 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     3121 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     3181 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     3241 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     3301 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     3361 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     3421 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     3481 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     3541 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     3601 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctt
     3661 ggtaccgagc tcggatccac tagtaacggc cgccagtgtg ctggaattcg cccttcaaaa
     3721 tctacgaaga taataaggtc tactattaca tttaataaat attcttattt aatttgagct
     3781 gtgtaaaatt agtgcgttta tccggggaat gtttatattt tatccgagga tttcccgaaa
     3841 ttgattaaaa aaatcaaatc tcttaaaaaa ttagaaacaa cctgttaaaa ataagcttat
     3901 agaaaaaatg taaagtgttg ctacaaggac aatcaattaa tcatcataac gaaaactttt
     3961 aggatcttga aaagcacgtc ccgagatggg aaagtgtgtt tcatttccag gcgatccttt
     4021 tcgcttcacg aaatttttga ttgctacctc gtttagagcc ctaagacaaa atacttcttc
     4081 aaaaaaaagc gaactcgaac ccaaactttg tactactatg ctaaaatgtc tgcatgcatg
     4141 ggatgtcgcg caaacgagtc caataagaaa taaaactgct cgaattcaac gtgacgctac
     4201 ccgcaatgct ctacccttaa ctttatacat cgctagaacc gcgttcgcat tcgctaacta
     4261 tataagcact ttcccggccc tacaacttgg gtcttcgaga agacctgttt tagagctatg
     4321 ctggaaacag catagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
     4381 ccgagtcggt gctttttttt tagggcgaat tctgcagata tccatcacac tggcggccgc
     4441 tcgagcatgc atctagaggg cccaattcgc cctatagtga gtcgtattac aattcactgg
     4501 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     4561 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgc
//
