LOCUS       sequence-348639-        7677 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of sgRNA under D. melanogaster U6-3 _(CR31539) promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7677)
  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 7677)
  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: pLib6.6B
FEATURES             Location/Qualifiers
     source          1..7677
                     /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        2594..2669
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2960..5469
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5C promoter"
     CDS             join(5501..5503,5504..5506,5507..6217)
                     /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(5546..5567)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             6221..6274
                     /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             6299..6898
                     /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(6299..6318)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6795..6815
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6906..7040
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6930..6949)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6984..7003
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    7234..7267
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     primer_bind     complement(7283..7300)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7283..7299)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7292..7314)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(7508..7527)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7627..7649
                     /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 acttcgaaaa tttttgaata gcccaggttg
     2341 ataaaaattc atttcatacg ttttataact tatgccccta agtatttttt gaccatagtg
     2401 tttcaattct acattaattt tacagagtag aatgaaacgc cacctactca gccaagaggc
     2461 gaaaaggtta gctcgccaag cagagagggc gccagtgctc actacttttt ataattctca
     2521 acttcttttt ccagactcag ttcgtatata tagacctatt ttcaatttaa cgtcggggtc
     2581 ttcgagaaga cctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
     2641 aacttgaaaa agtggcaccg agtcggtgct tttttgccta cctggagcct gagagttgtt
     2701 caataaaata aaaatgtttc gtttttttgc tttcgccagt atttattatt tttcatcaat
     2761 atgtattcaa tttggtatgt atttagtaat tgtaatatat agacaatggt tttccgttga
     2821 cgtacataca tctgacgtgt gtttatttag acataatagt tatgttttca catcttttta
     2881 atgttcgctt aatgcgtatg cggtacccag catgcaattc tatattctaa aaacacaaat
     2941 gatacttcta aaaaaaaatc atgaatggca tcaactctga atcaaatctt tgcagatgca
     3001 cctacttctc atttccactg tcacatcatt tttccagatc tcgctgcctg ttatgtggcc
     3061 cacaaaccaa gacacgtttt atggccatta aagctggctg atcgtcgcca aacaccaaat
     3121 acataatgaa tatgtacaca ttcgagaaag aagcgatcaa agaagcatct tcgggcggag
     3181 taggagaatg cggaggagaa ggagaacgag ctgatctagt atctctccac aatccaatgc
     3241 caactgacca actggccata ttcggagcaa tttgaagcca atttccatcg cctggcgatc
     3301 gctccattct tggctatatg tttttcaccg ttacccgggg ccattttcaa agactcgtcg
     3361 gcaagataag attgtgtcac tcgctgtctc tcttcatttg tcgaagaatg ctgaggaatt
     3421 tcgcgatgac gtcggcgagt attttgaaga atgagaataa tttgtattta tacgaaaatc
     3481 agttagtgga attttctaca aaaacatgtt atctatagat aattttgttg caaaatatgt
     3541 tgactatgac aaagattgta tgtatatacc tttaatgtat tctcattttc ttatgtattt
     3601 ataatggcaa tgatgatact gatgatattt taagatgatg ccagaccaaa aggcttgaat
     3661 ttctgcgtct tttgccgaac gcagtgcatg tgcaattgtt gttttttgga atattcaatt
     3721 ttcggactgt ccgctttgat ttcagtttct tggcttattc aaaaagcaaa gtaaagccaa
     3781 aaaagcgaga tggcaatacc aaatgcggca aaacggtagt ggaaggaaag gggtgcgggg
     3841 cagcggaagg aagggtgggg cggggcgtgg cggggtctgt ggctgggcgc gacgtcaccg
     3901 acgttggagc cactcctttg accatgtgtg cgtgtgtgta ttattcgtgt ctcgccactc
     3961 gccggttgtt tttttctttt tatgctgcgc tctctctagc gccatctcgc ttacgcatgc
     4021 tcaacgcacc gcatgttgcc gtttcctttt atgcgtcatt ttggctcgaa ataggcaatt
     4081 atttaaacaa agattagtca acgaaaacgc taaaataaat aagtctacaa tatggttact
     4141 tattgccatg tgtgtgcagc caacgatagc aacaaaagca acaacacagg tggctttccc
     4201 tctttcactt tttgtttgca agccgcgtgc gagcaagacg gcacgaccgg caaacgcaat
     4261 tacgctgaca aagagcagac gaagttttgg cgaaaaacat caaggcgcct gatacgaatg
     4321 catttgcaat aacaattgcg atatttaata ttgtttatga agctgtttga cttcaaaaca
     4381 cacaaaaaaa aaaataaaac aaattatttg aaagagaatt aggaatcgga cgcttatcgt
     4441 tagggtaaca acaagaaatg cttactgagt cacagcctct ggaaaactgc cgcaagccag
     4501 agagagagag aaaaagaggg agagcagctt agaccgcatg tgcttgtgtg tgaggcgtct
     4561 ctctcttcgt ctctgttgcg caaacgcata gactgcactg aaaaaatcga ttacctattt
     4621 tttatgaatg aatatttgca ctattactat tcaaaactat taagatagca atcacattca
     4681 atagccaaat actataccac ctgagcgatg caacgaaatg atcaatttga gcaaaaatgc
     4741 tgcatattta ggacggcatc attatagaaa tgcttcttgc tgtgtacttt tctctcgtct
     4801 ggcagctgtt tcgccgttat tgttaaaacc ggcttaagtt aggtgtgttt tctacgacta
     4861 gtgaatgccc tactagaaga tgtgtgttgc acaaaatgtc cctggaataa ccaatttgaa
     4921 gtgcagatag cagtaaacgt aagctaatat gaatattatt taactgtaat gttttaatat
     4981 cgctggacat tactaataaa cccactataa acacatgtac atatgtatgt tttggcatac
     5041 aatgagtagt tggggaaaaa atgtgtaaaa gcaccgtgac catcacagca taaagataac
     5101 cagctgaagt atcgaatatg agtaaccccc aaattgaatc acatgccgca actgatagga
     5161 cccatggaag tacactcttc atggcgatat acaagacaca cacaagcacg aacacccagt
     5221 tgcggaggaa attctccgta aatgaaaacc caatcggcga acaattcata cccatatatg
     5281 gtaaaagttt tgaacgcgac ttgagagcgg agagcattgc ggctgataag gttttagcgc
     5341 taagcgggct ttataaaacg ggctgcggga ccagttttca tatcactacc gtttgagttc
     5401 ttgtgctgtg tggatactcc tcccgacaca aagccgctcc atcagccagc agtcgtctaa
     5461 tccagagacc ccggatccag gaattcgccc ttgtttaaac atggtgagca agggcgagga
     5521 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     5581 gttcagcgtg aggggcgagg gcgagggcga tgccaccaac ggcaagctga ccctgaagtt
     5641 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgagcca
     5701 cggcgtgcag tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     5761 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaccta
     5821 caagacccgc gccgaggtga agttcgaggg cgacacccta gtgaaccgca tcgagctgaa
     5881 gggcgtcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaacttcaa
     5941 cagccacaac atctatatca tggccgtcaa gcagaagaac ggcatcaagg tgaacttcaa
     6001 gatccgccac aacgtggagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     6061 ccccatcggc gacggccccg tgctgctgcc cgacagccac tacctgagca cccagtccgt
     6121 gctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tccgcaccgc
     6181 cgccgggatc actctcggca tggacgagct gtacaagctt gagggcagag gaagccttct
     6241 aacatgcggt gacgtggagg agaatcccgg ccctgctagc ggtagcggca gcggtagcat
     6301 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc gggccgtacg
     6361 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg acccggaccg
     6421 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     6481 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     6541 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     6601 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     6661 cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag
     6721 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     6781 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     6841 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgact
     6901 cgagggatcc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     6961 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     7021 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     7081 gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt atggctgatt
     7141 atgatcagtc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg
     7201 cggggcatga ctatcgtcgc cgcacttatg gtacgcgccc ggggagccca agggcacgcc
     7261 ctggcacccg aagggcgaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     7321 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     7381 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc
     7441 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca
     7501 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     7561 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     7621 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcga
//
