LOCUS       sequence-333376-        6431 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 6431)
  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 6431)
  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: pVMG0217_Cq-U6-2b_2xBbsI-gRNA
FEATURES             Location/Qualifiers
     source          1..6431
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      14..442
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(101..120)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     311..332
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             786..1580
                     /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(840..859)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1450..1469
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(1598..1666,1667..2458)
                     /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(1816..1835)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2629..3217
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3118..3137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3371..3388
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3505..3526
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3541..3546,3547..3564,3565..3571)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3579..3595
                     /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     3584..3606
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3603..3619
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3603..3619
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_RNA        4986..5061
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(6262..6281)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6263..6281)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6288..6305)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6288..6304)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6297..6319)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
       61 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
      121 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
      181 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
      241 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
      301 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
      361 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
      421 taacaaaaat ttaacgcgaa ttttaacaaa attcagggcg caagggctgc taaaggaagc
      481 ggaacacgta gaaagccagt ccgcagaaac ggtgctgacc ccggatgaat gtcagctact
      541 gggctatctg gacaagggaa aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc
      601 ttacatggcg atagctagac tgggcggttt tatggacagc aagcgaaccg gaattgccag
      661 ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt aaactggatg gctttcttgc
      721 cgccaaggat ctgatggcgc aggggatcaa gatctgatca agagacagga tgaggatcgt
      781 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
      841 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
      901 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
      961 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     1021 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     1081 ggcaggatct cctgtcatcc caccttgctc ctgccgagaa agtatccatc atggctgatg
     1141 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     1201 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     1261 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     1321 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     1381 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     1441 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     1501 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     1561 ttcttgacga gttcttctga attgaaaaag gaagagtatg agtattcaac atttccgtgt
     1621 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     1681 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     1741 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     1801 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     1861 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     1921 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     1981 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     2041 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     2101 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     2161 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     2221 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     2281 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     2341 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     2401 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     2461 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     2521 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     2581 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     2641 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     2701 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     2761 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     2821 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     2881 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     2941 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3001 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3061 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     3121 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     3181 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     3241 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     3301 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     3361 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     3421 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     3481 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
     3541 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
     3601 cacaggaaac agctatgacc atgattacgc caagcttggt accgagctcg gatccactag
     3661 taacggccgc cagtgtgctg gaattcgccc ttgccgaagc aaagacaagt tcggagggtg
     3721 ccctcgttgc ttatatagtt cgtgaatcgt taaacggttc tagcgatggc ggagtgcgga
     3781 gtgcgaagtg agcgaaagaa acgcgaaaaa gtgagaacat ttgtaaggtt cacagcaatg
     3841 gggggcaggt aaacgtttag taatcgatta cccgtcagtt cgaggttagg ttatgtacct
     3901 gcgattgaat ttaaaacaag atttcgctaa ttgaatcaga attgccgttt gtgactaaat
     3961 gcgactctca aaatatgcag attaacagtc tggacccgga gcctaatttt tctgttacat
     4021 tcttattgga attccatata aactgtaacg ggaactgcag gcaccgggtc ctgactatta
     4081 atctcagtcg acgcgtctgc tccgcaacaa aatgcatagc aaatcaaatt tgtctggcat
     4141 tttcaattat acccattacg actcgcagtg cttcaacaac tcctaaataa ccgtaagcgg
     4201 ttttactccc cttccctccg ttgtctggag taaaaacgag gcgtcagtaa tgggggactg
     4261 cccccgccct ccatcgtaac tcccctcaag caacggccca aatcatttaa tcccacacaa
     4321 tttgtttcaa tttttcttct tgcaagtcgt cgtcgtcgtc tggcaatgag aatctcgcga
     4381 gataagatgc ctgcttctgc tcatcagcat gatcatcatc actggcgagg aaggaggctc
     4441 atttccttaa actcgtggga ggacgacact gcaatttgaa ggcgagagga ggttatgtta
     4501 cttttttccg ggcccttgcc ctaatgggtt ctgaatccct ctgtctgtgg ttgttgttgt
     4561 tgttcttggg ggtcgatgtc gtcgagccca ctccttgccc ggcctgtttg tgtgtatgtt
     4621 tatgtgataa aatattaatg atgcaacccc ggtggcagac actgctggcg aatgttgcgg
     4681 accagttaag gttctggtag acagggtgac gagcgctccg gttgacattg cagcatttta
     4741 ggggtggttc agagttctgg aaaaaagttt tataaataaa attccaattg gcatgaaatc
     4801 gacgttgcaa atggttttga gacagatcta ataggactta tttttaatat tgcaggtaat
     4861 ttacctgtta ttacctgtgc attgaacttg gaaacaaata ttaaacatcg ctagaactgt
     4921 tttgaagtgc gctaactata taagcaaatt ttgagccctt tgtactcggg tcttcgagaa
     4981 gacctgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
     5041 aaagtggcac cgagtcggtg cttttttttt gattctggtt tatttttgtt ctcttttgga
     5101 agataagatt ttttaaaatt aaaaacctac ttttttacga atcgtaaaaa ttttattgaa
     5161 aataatggtt cagaacatta tattaatgtt tatcgaaaat ctggcagcac tgccataagt
     5221 aatgagcact tgaaaaaaat gtatataact tgtcaacgtt atcagtcttg atgaaaattt
     5281 aattcgcttt ttgtacacta ttatttaaga aaacattttc agtatacact catccctcat
     5341 attcggaaca gtttacagat cggccaatgt ttaataaatc atataaaatc gataattgaa
     5401 cataatgatt tgcttttacc ttcatgtgaa agcttttctt gcgatctttc gattgatgta
     5461 tagaccggct gtacattttt acgatttata tcaagttttc gaagaaaaac attgaccatt
     5521 gaaattcaca aattcggaac acttttttct tacgatgtaa acaaactttt ttttctctcc
     5581 tggtgtacat atttttttgc aaaataatga ctttgcactc tgaaaccaat agaactcatg
     5641 gttagtgatg ttttatgaat ggtctgataa cttttttgtg aaaatatcag ttaaattcag
     5701 gtgttccaca attgtgggag acacaataac atcccacaat catggaacgg ccaatttaca
     5761 ggcagtattt tgctgatctg gataaaatag tcttgaaatg cagtttttgt ttgaaaagta
     5821 ttacttttac tggtgaaata gcaagagaat gtccaaataa aggccttaaa aatagcaggg
     5881 tcggcagaaa agttgcattt tgcattctat tgacaaatta ccacaaaagt gttccgaatt
     5941 tgtgggtgtt ccgaatatgt gggatgactg taccattagg tttggcaact ctgcttaagg
     6001 gtgcacatca accaaggaag tttttctttt cttattccaa aatagtcaaa ctttgttgta
     6061 aataactttg tagacagtac ttaatgggat gaataaaaaa tcgtatcgaa agtacccgta
     6121 gttttgaaga tactaaaatg tctgtaataa aaatcttggt gcaaaagctc agattgatgt
     6181 gcaccgtaaa acaagggcga attctgcaga tatccatcac actggcggcc gctcgagcat
     6241 gcatctagag ggcccaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt
     6301 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     6361 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     6421 ttgcgcagcc t
//
