LOCUS       sequence-348535-        7570 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of sgRNA under C. quinquefasciatus U6 (CPIJ039728) 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7570)
  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 7570)
  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-Cquin_728
FEATURES             Location/Qualifiers
     source          1..7570
                     /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        2515..2590
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2853..5362
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5C promoter"
     CDS             join(5394..5396,5397..5399,5400..6110)
                     /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(5439..5460)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             6114..6167
                     /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             6192..6791
                     /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(6192..6211)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6688..6708
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6799..6933
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6823..6842)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6877..6896
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    7127..7160
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     primer_bind     complement(7176..7193)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7176..7192)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7185..7207)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(7401..7420)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7520..7542
                     /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 acttcgaagg catgaaatcg acgttgcaaa
     2341 ttgttttgag acagatctaa taggacttct ttttgatatt gcaggtaatt tacctgttat
     2401 tacctgtgca ttgaacttgg aaacaaatat taaatatcgc tagaactgaa ttgaagtgct
     2461 ataactatat aagcaaaatt tgagcccacg gtactcgggt cttcgagaag acctgtttta
     2521 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
     2581 gagtcggtgc ttttttttgt gattctggtt tctttttgtt ttcttttgga agattagatt
     2641 tttttatttt ttgcctacct ggagcctgag agttgttcaa taaaataaaa atgtttcgtt
     2701 tttttgcttt cgccagtatt tattattttt catcaatatg tattcaattt ggtatgtatt
     2761 tagtaattgt aatatataga caatggtttt ccgtggtacc cagcatgcaa ttctatattc
     2821 taaaaacaca aatgatactt ctaaaaaaaa atcatgaatg gcatcaactc tgaatcaaat
     2881 ctttgcagat gcacctactt ctcatttcca ctgtcacatc atttttccag atctcgctgc
     2941 ctgttatgtg gcccacaaac caagacacgt tttatggcca ttaaagctgg ctgatcgtcg
     3001 ccaaacacca aatacataat gaatatgtac acattcgaga aagaagcgat caaagaagca
     3061 tcttcgggcg gagtaggaga atgcggagga gaaggagaac gagctgatct agtatctctc
     3121 cacaatccaa tgccaactga ccaactggcc atattcggag caatttgaag ccaatttcca
     3181 tcgcctggcg atcgctccat tcttggctat atgtttttca ccgttacccg gggccatttt
     3241 caaagactcg tcggcaagat aagattgtgt cactcgctgt ctctcttcat ttgtcgaaga
     3301 atgctgagga atttcgcgat gacgtcggcg agtattttga agaatgagaa taatttgtat
     3361 ttatacgaaa atcagttagt ggaattttct acaaaaacat gttatctata gataattttg
     3421 ttgcaaaata tgttgactat gacaaagatt gtatgtatat acctttaatg tattctcatt
     3481 ttcttatgta tttataatgg caatgatgat actgatgata ttttaagatg atgccagacc
     3541 aaaaggcttg aatttctgcg tcttttgccg aacgcagtgc atgtgcaatt gttgtttttt
     3601 ggaatattca attttcggac tgtccgcttt gatttcagtt tcttggctta ttcaaaaagc
     3661 aaagtaaagc caaaaaagcg agatggcaat accaaatgcg gcaaaacggt agtggaagga
     3721 aaggggtgcg gggcagcgga aggaagggtg gggcggggcg tggcggggtc tgtggctggg
     3781 cgcgacgtca ccgacgttgg agccactcct ttgaccatgt gtgcgtgtgt gtattattcg
     3841 tgtctcgcca ctcgccggtt gtttttttct ttttatgctg cgctctctct agcgccatct
     3901 cgcttacgca tgctcaacgc accgcatgtt gccgtttcct tttatgcgtc attttggctc
     3961 gaaataggca attatttaaa caaagattag tcaacgaaaa cgctaaaata aataagtcta
     4021 caatatggtt acttattgcc atgtgtgtgc agccaacgat agcaacaaaa gcaacaacac
     4081 aggtggcttt ccctctttca ctttttgttt gcaagccgcg tgcgagcaag acggcacgac
     4141 cggcaaacgc aattacgctg acaaagagca gacgaagttt tggcgaaaaa catcaaggcg
     4201 cctgatacga atgcatttgc aataacaatt gcgatattta atattgttta tgaagctgtt
     4261 tgacttcaaa acacacaaaa aaaaaaataa aacaaattat ttgaaagaga attaggaatc
     4321 ggacgcttat cgttagggta acaacaagaa atgcttactg agtcacagcc tctggaaaac
     4381 tgccgcaagc cagagagaga gagaaaaaga gggagagcag cttagaccgc atgtgcttgt
     4441 gtgtgaggcg tctctctctt cgtctctgtt gcgcaaacgc atagactgca ctgaaaaaat
     4501 cgattaccta ttttttatga atgaatattt gcactattac tattcaaaac tattaagata
     4561 gcaatcacat tcaatagcca aatactatac cacctgagcg atgcaacgaa atgatcaatt
     4621 tgagcaaaaa tgctgcatat ttaggacggc atcattatag aaatgcttct tgctgtgtac
     4681 ttttctctcg tctggcagct gtttcgccgt tattgttaaa accggcttaa gttaggtgtg
     4741 ttttctacga ctagtgaatg ccctactaga agatgtgtgt tgcacaaaat gtccctggaa
     4801 taaccaattt gaagtgcaga tagcagtaaa cgtaagctaa tatgaatatt atttaactgt
     4861 aatgttttaa tatcgctgga cattactaat aaacccacta taaacacatg tacatatgta
     4921 tgttttggca tacaatgagt agttggggaa aaaatgtgta aaagcaccgt gaccatcaca
     4981 gcataaagat aaccagctga agtatcgaat atgagtaacc cccaaattga atcacatgcc
     5041 gcaactgata ggacccatgg aagtacactc ttcatggcga tatacaagac acacacaagc
     5101 acgaacaccc agttgcggag gaaattctcc gtaaatgaaa acccaatcgg cgaacaattc
     5161 atacccatat atggtaaaag ttttgaacgc gacttgagag cggagagcat tgcggctgat
     5221 aaggttttag cgctaagcgg gctttataaa acgggctgcg ggaccagttt tcatatcact
     5281 accgtttgag ttcttgtgct gtgtggatac tcctcccgac acaaagccgc tccatcagcc
     5341 agcagtcgtc taatccagag accccggatc caggaattcg cccttgttta aacatggtga
     5401 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     5461 taaacggcca caagttcagc gtgaggggcg agggcgaggg cgatgccacc aacggcaagc
     5521 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     5581 ccaccctgag ccacggcgtg cagtgcttcg cccgctaccc cgaccacatg aagcagcacg
     5641 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     5701 acgacggcac ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctagtgaacc
     5761 gcatcgagct gaagggcgtc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     5821 agtacaactt caacagccac aacatctata tcatggccgt caagcagaag aacggcatca
     5881 aggtgaactt caagatccgc cacaacgtgg aggacggcag cgtgcagctc gccgaccact
     5941 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacagc cactacctga
     6001 gcacccagtc cgtgctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     6061 agttccgcac cgccgccggg atcactctcg gcatggacga gctgtacaag cttgagggca
     6121 gaggaagcct tctaacatgc ggtgacgtgg aggagaatcc cggccctgct agcggtagcg
     6181 gcagcggtag catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc
     6241 cccgggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg
     6301 tcgacccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg
     6361 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga
     6421 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg
     6481 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc
     6541 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca
     6601 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc
     6661 ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca
     6721 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc
     6781 ccggtgcctg actcgaggga tccagacatg ataagataca ttgatgagtt tggacaaacc
     6841 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     6901 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     6961 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     7021 ggtatggctg attatgatca gtcgaccgat gcccttgaga gccttcaacc cagtcagctc
     7081 cttccggtgg gcgcggggca tgactatcgt cgccgcactt atggtacgcg cccggggagc
     7141 ccaagggcac gccctggcac ccgaagggcg aattcactgg ccgtcgtttt acaacgtcgt
     7201 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     7261 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     7321 aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     7381 cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     7441 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     7501 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     7561 aaacgcgcga
//
