LOCUS       sequence-348666-        7460 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of sgRNA under mosquito consensus U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7460)
  AUTHORS   Viswanatha R, Mameli E, Rodiger J, Merckaert P, Feitosa-Suntheimer 
            F, Colpitts TM, Mohr SE, Hu Y, Perrimon N
  TITLE     Bioinformatic and cell-based tools for pooled CRISPR knockout 
            screening in mosquitos.
  JOURNAL   Nat Commun. 2021 Nov 24;12(1):6825. doi: 10.1038/s41467-021-27129-3.
  PUBMED    34819517
REFERENCE   2  (bases 1 to 7460)
  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: "2021-11-24"; volume: "12(1):6825. doi"; pages: " 
            10.1038/s41467-021-27129-3"
COMMENT     Sequence Label: pLib6.4B-Consensus
FEATURES             Location/Qualifiers
     source          1..7460
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /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(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1721..1740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1974..1991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2144..2149,2150..2167,2168..2174)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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     2187..2209
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2206..2222
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    2276..2309
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     misc_RNA        2436..2511
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2743..5252
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5C promoter"
     CDS             join(5284..5286,5287..5289,5290..6000)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(5329..5350)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             6004..6057
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             6082..6681
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(6082..6101)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6578..6598
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6689..6823
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6713..6732)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6767..6786
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    7017..7050
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     primer_bind     complement(7066..7083)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7066..7082)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7075..7097)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(7291..7310)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7410..7432
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     2221 accatgatta cgccaagctt gcatgcctgc aggtcgactc tagaggatcc cccgggtgcc
     2281 agggcgtgcc cttgggctcc ccgggcgcgt acttcgaatt cccactattc aatcacttaa
     2341 aaatataagt ttatccatcg ctagaaccgt tttgacgtgc gctaactata taagcaaaat
     2401 ggtttccctc tatacttggg tcttcgagaa gacctgtttt agagctagaa atagcaagtt
     2461 aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttttt
     2521 tttgcctacc tggagcctga gagttgttca ataaaataaa aatgtttcgt ttttttgctt
     2581 tcgccagtat ttattatttt tcatcaatat gtattcaatt tggtatgtat ttagtaattg
     2641 taatatatag acaatggttt tccgttgacg tacatacatc tgacggtacc cagcatgcaa
     2701 ttctatattc taaaaacaca aatgatactt ctaaaaaaaa atcatgaatg gcatcaactc
     2761 tgaatcaaat ctttgcagat gcacctactt ctcatttcca ctgtcacatc atttttccag
     2821 atctcgctgc ctgttatgtg gcccacaaac caagacacgt tttatggcca ttaaagctgg
     2881 ctgatcgtcg ccaaacacca aatacataat gaatatgtac acattcgaga aagaagcgat
     2941 caaagaagca tcttcgggcg gagtaggaga atgcggagga gaaggagaac gagctgatct
     3001 agtatctctc cacaatccaa tgccaactga ccaactggcc atattcggag caatttgaag
     3061 ccaatttcca tcgcctggcg atcgctccat tcttggctat atgtttttca ccgttacccg
     3121 gggccatttt caaagactcg tcggcaagat aagattgtgt cactcgctgt ctctcttcat
     3181 ttgtcgaaga atgctgagga atttcgcgat gacgtcggcg agtattttga agaatgagaa
     3241 taatttgtat ttatacgaaa atcagttagt ggaattttct acaaaaacat gttatctata
     3301 gataattttg ttgcaaaata tgttgactat gacaaagatt gtatgtatat acctttaatg
     3361 tattctcatt ttcttatgta tttataatgg caatgatgat actgatgata ttttaagatg
     3421 atgccagacc aaaaggcttg aatttctgcg tcttttgccg aacgcagtgc atgtgcaatt
     3481 gttgtttttt ggaatattca attttcggac tgtccgcttt gatttcagtt tcttggctta
     3541 ttcaaaaagc aaagtaaagc caaaaaagcg agatggcaat accaaatgcg gcaaaacggt
     3601 agtggaagga aaggggtgcg gggcagcgga aggaagggtg gggcggggcg tggcggggtc
     3661 tgtggctggg cgcgacgtca ccgacgttgg agccactcct ttgaccatgt gtgcgtgtgt
     3721 gtattattcg tgtctcgcca ctcgccggtt gtttttttct ttttatgctg cgctctctct
     3781 agcgccatct cgcttacgca tgctcaacgc accgcatgtt gccgtttcct tttatgcgtc
     3841 attttggctc gaaataggca attatttaaa caaagattag tcaacgaaaa cgctaaaata
     3901 aataagtcta caatatggtt acttattgcc atgtgtgtgc agccaacgat agcaacaaaa
     3961 gcaacaacac aggtggcttt ccctctttca ctttttgttt gcaagccgcg tgcgagcaag
     4021 acggcacgac cggcaaacgc aattacgctg acaaagagca gacgaagttt tggcgaaaaa
     4081 catcaaggcg cctgatacga atgcatttgc aataacaatt gcgatattta atattgttta
     4141 tgaagctgtt tgacttcaaa acacacaaaa aaaaaaataa aacaaattat ttgaaagaga
     4201 attaggaatc ggacgcttat cgttagggta acaacaagaa atgcttactg agtcacagcc
     4261 tctggaaaac tgccgcaagc cagagagaga gagaaaaaga gggagagcag cttagaccgc
     4321 atgtgcttgt gtgtgaggcg tctctctctt cgtctctgtt gcgcaaacgc atagactgca
     4381 ctgaaaaaat cgattaccta ttttttatga atgaatattt gcactattac tattcaaaac
     4441 tattaagata gcaatcacat tcaatagcca aatactatac cacctgagcg atgcaacgaa
     4501 atgatcaatt tgagcaaaaa tgctgcatat ttaggacggc atcattatag aaatgcttct
     4561 tgctgtgtac ttttctctcg tctggcagct gtttcgccgt tattgttaaa accggcttaa
     4621 gttaggtgtg ttttctacga ctagtgaatg ccctactaga agatgtgtgt tgcacaaaat
     4681 gtccctggaa taaccaattt gaagtgcaga tagcagtaaa cgtaagctaa tatgaatatt
     4741 atttaactgt aatgttttaa tatcgctgga cattactaat aaacccacta taaacacatg
     4801 tacatatgta tgttttggca tacaatgagt agttggggaa aaaatgtgta aaagcaccgt
     4861 gaccatcaca gcataaagat aaccagctga agtatcgaat atgagtaacc cccaaattga
     4921 atcacatgcc gcaactgata ggacccatgg aagtacactc ttcatggcga tatacaagac
     4981 acacacaagc acgaacaccc agttgcggag gaaattctcc gtaaatgaaa acccaatcgg
     5041 cgaacaattc atacccatat atggtaaaag ttttgaacgc gacttgagag cggagagcat
     5101 tgcggctgat aaggttttag cgctaagcgg gctttataaa acgggctgcg ggaccagttt
     5161 tcatatcact accgtttgag ttcttgtgct gtgtggatac tcctcccgac acaaagccgc
     5221 tccatcagcc agcagtcgtc taatccagag accccggatc caggaattcg cccttgttta
     5281 aacatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     5341 gacggcgacg taaacggcca caagttcagc gtgaggggcg agggcgaggg cgatgccacc
     5401 aacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     5461 accctcgtga ccaccctgag ccacggcgtg cagtgcttcg cccgctaccc cgaccacatg
     5521 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     5581 ttcttcaagg acgacggcac ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     5641 ctagtgaacc gcatcgagct gaagggcgtc gacttcaagg aggacggcaa catcctgggg
     5701 cacaagctgg agtacaactt caacagccac aacatctata tcatggccgt caagcagaag
     5761 aacggcatca aggtgaactt caagatccgc cacaacgtgg aggacggcag cgtgcagctc
     5821 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacagc
     5881 cactacctga gcacccagtc cgtgctgagc aaagacccca acgagaagcg cgatcacatg
     5941 gtcctgctgg agttccgcac cgccgccggg atcactctcg gcatggacga gctgtacaag
     6001 cttgagggca gaggaagcct tctaacatgc ggtgacgtgg aggagaatcc cggccctgct
     6061 agcggtagcg gcagcggtag catgaccgag tacaagccca cggtgcgcct cgccacccgc
     6121 gacgacgtcc cccgggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
     6181 cgccacaccg tcgacccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
     6241 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg
     6301 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
     6361 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg
     6421 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac
     6481 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc
     6541 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg
     6601 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
     6661 acccgcaagc ccggtgcctg actcgaggga tccagacatg ataagataca ttgatgagtt
     6721 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     6781 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     6841 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct
     6901 ctacaaatgt ggtatggctg attatgatca gtcgaccgat gcccttgaga gccttcaacc
     6961 cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt atggtacgcg
     7021 cccggggagc ccaagggcac gccctggcac ccgaagggcg aattcactgg ccgtcgtttt
     7081 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     7141 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     7201 gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     7261 tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     7321 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     7381 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     7441 gtcatcaccg aaacgcgcga
//
