LOCUS       sequence-333388-        4810 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 4810)
  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 4810)
  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: pVMG0146_Cq-U6-1_2xBbsI-gRNA
FEATURES             Location/Qualifiers
     source          1..4810
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    10..26
                     /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     15..37
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     34..50
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     34..50
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_RNA        complement(251..326)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1072..1091)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1073..1091)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1098..1115)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1098..1114)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1107..1129)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1255..1683
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1342..1361)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1552..1573
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             2027..2821
                     /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(2081..2100)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2691..2710
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(2839..2907,2908..3699)
                     /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(3057..3076)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3870..4458
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4359..4378
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4612..4629
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4746..4767
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4782..4787,4788..4805,4806..4810,1..2)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
       61 ccaagcttgg taccgagctc ggatccacta gtaacggccg ccagtgtgct ggaattcgcc
      121 cttaaggaac acctgttccc tctgcgaggg agcaccggtg aaaaatttgt aaaacattct
      181 ggttcaagtt caaagtgatt taagaaaatc cgattttcgg aatgttgtta ctcatccgcc
      241 accaaaaaaa gcaccgactc ggtgccactt tttcaagttg ataacggact agccttattt
      301 taacttgcta tttctagctc taaaacaggt cttctcgaag acccaagttc ggagggtgcc
      361 ctcgttgctt atatagttcg tgaatcgtta aacggttcta gcgatggcgg agtgcggagt
      421 gcgaagtgag cgaaagaaac gcgaaaaagt gagaacattt gtaaggttca cagcaatggg
      481 gggcaggtaa acgtttagta atcgattacc cgtcagttcg aggttaggtt atgtacctgc
      541 gattgaattt aaaacaagat ttcgctaatt gaatcagaat tgccgtttgt gactaaatgc
      601 gactctcaaa atatgcagat taacagtctg gacccggagc ctaatttttc tgttacattc
      661 ttattggaat tccatataaa ctgtaacggg aactgcaggc accgggtcct gactattaat
      721 ctcagtcgac gcgtctgctc cgcaacaaaa tgcatagcaa atcaaatttg tctggcattt
      781 tcaattatac ccattacgac tcgcagtgct tcaacaactc ctaaataacc gtaagcggtt
      841 ttactcccct tccctccgtt gtctggagta aaaacgaggc gtcagtaatg ggggactgcc
      901 cccgccctcc atcgtaactc ccctcaagca acggcccaaa tcatttaatc ccacacaatt
      961 tgtttcaatt tttcttcttg caagtcgtcg tcgtcgtctg ggaagggcga attctgcaga
     1021 tatccatcac actggcggcc gctcgagcat gcatctagag ggcccaattc gccctatagt
     1081 gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     1141 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     1201 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc
     1261 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     1321 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     1381 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     1441 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     1501 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     1561 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     1621 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     1681 attttaacaa aattcagggc gcaagggctg ctaaaggaag cggaacacgt agaaagccag
     1741 tccgcagaaa cggtgctgac cccggatgaa tgtcagctac tgggctatct ggacaaggga
     1801 aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc gatagctaga
     1861 ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc cctctggtaa
     1921 ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga tctgatggcg
     1981 caggggatca agatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     2041 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     2101 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     2161 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     2221 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     2281 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     2341 ccaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     2401 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     2461 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     2521 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     2581 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     2641 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     2701 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     2761 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     2821 aattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     2881 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     2941 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     3001 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     3061 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     3121 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     3181 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     3241 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     3301 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     3361 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     3421 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     3481 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     3541 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     3601 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     3661 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     3721 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     3781 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     3841 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     3901 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     3961 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     4021 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     4081 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     4141 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     4201 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     4261 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     4321 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     4381 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     4441 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     4501 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     4561 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     4621 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     4681 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     4741 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     4801 gctcgtatgt
//
