LOCUS       sequence-348636-        7682 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of sgRNA under D. melanogaster U6-2 _(_CR32867) promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7682)
  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 7682)
  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
FEATURES             Location/Qualifiers
     source          1..7682
                     /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)"
     promoter        2319..2718
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
     misc_RNA        2741..2816
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2965..5474
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5C promoter"
     CDS             join(5506..5508,5509..5511,5512..6222)
                     /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(5551..5572)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             6226..6279
                     /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             6304..6903
                     /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(6304..6323)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6800..6820
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6911..7045
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6935..6954)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6989..7008
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    7239..7272
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     primer_bind     complement(7288..7305)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7288..7304)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7297..7319)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(7513..7532)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7632..7654
                     /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 acttcgaagt tcgacttgca gcctgaaata
     2341 cggcacgagt aggaaaagcc gagtcaaatg ccgaatgcag agtctcatta cagcacaatc
     2401 aactcaagaa aaactcgaca cttttttacc atttgcactt aaatcctttt ttattcgtta
     2461 tgtatacttt ttttggtccc taaccaaaac aaaaccaaac tctcttagtc gtgcctctat
     2521 atttaaaact atcaatttat tatagtcaat aaatcgaact gtgttttcaa caaacgaaca
     2581 ataggacact ttgattctaa aggaaatttt gaaaatctta agcagagggt tcttaagacc
     2641 atttgccaat tcttataatt ctcaactgct ctttcctgat gttgttcatt tatataggta
     2701 tgttttcctc aatacttcgt tcgggtcttc gagaagacct gttttagagc tagaaatagc
     2761 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     2821 ttgttttaga gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc
     2881 caattctgca gacaaatggc tctagaggta cccagcatgc aattctatat tctaaaaaca
     2941 caaatgatac ttctaaaaaa aaatcatgaa tggcatcaac tctgaatcaa atctttgcag
     3001 atgcacctac ttctcatttc cactgtcaca tcatttttcc agatctcgct gcctgttatg
     3061 tggcccacaa accaagacac gttttatggc cattaaagct ggctgatcgt cgccaaacac
     3121 caaatacata atgaatatgt acacattcga gaaagaagcg atcaaagaag catcttcggg
     3181 cggagtagga gaatgcggag gagaaggaga acgagctgat ctagtatctc tccacaatcc
     3241 aatgccaact gaccaactgg ccatattcgg agcaatttga agccaatttc catcgcctgg
     3301 cgatcgctcc attcttggct atatgttttt caccgttacc cggggccatt ttcaaagact
     3361 cgtcggcaag ataagattgt gtcactcgct gtctctcttc atttgtcgaa gaatgctgag
     3421 gaatttcgcg atgacgtcgg cgagtatttt gaagaatgag aataatttgt atttatacga
     3481 aaatcagtta gtggaatttt ctacaaaaac atgttatcta tagataattt tgttgcaaaa
     3541 tatgttgact atgacaaaga ttgtatgtat atacctttaa tgtattctca ttttcttatg
     3601 tatttataat ggcaatgatg atactgatga tattttaaga tgatgccaga ccaaaaggct
     3661 tgaatttctg cgtcttttgc cgaacgcagt gcatgtgcaa ttgttgtttt ttggaatatt
     3721 caattttcgg actgtccgct ttgatttcag tttcttggct tattcaaaaa gcaaagtaaa
     3781 gccaaaaaag cgagatggca ataccaaatg cggcaaaacg gtagtggaag gaaaggggtg
     3841 cggggcagcg gaaggaaggg tggggcgggg cgtggcgggg tctgtggctg ggcgcgacgt
     3901 caccgacgtt ggagccactc ctttgaccat gtgtgcgtgt gtgtattatt cgtgtctcgc
     3961 cactcgccgg ttgttttttt ctttttatgc tgcgctctct ctagcgccat ctcgcttacg
     4021 catgctcaac gcaccgcatg ttgccgtttc cttttatgcg tcattttggc tcgaaatagg
     4081 caattattta aacaaagatt agtcaacgaa aacgctaaaa taaataagtc tacaatatgg
     4141 ttacttattg ccatgtgtgt gcagccaacg atagcaacaa aagcaacaac acaggtggct
     4201 ttccctcttt cactttttgt ttgcaagccg cgtgcgagca agacggcacg accggcaaac
     4261 gcaattacgc tgacaaagag cagacgaagt tttggcgaaa aacatcaagg cgcctgatac
     4321 gaatgcattt gcaataacaa ttgcgatatt taatattgtt tatgaagctg tttgacttca
     4381 aaacacacaa aaaaaaaaat aaaacaaatt atttgaaaga gaattaggaa tcggacgctt
     4441 atcgttaggg taacaacaag aaatgcttac tgagtcacag cctctggaaa actgccgcaa
     4501 gccagagaga gagagaaaaa gagggagagc agcttagacc gcatgtgctt gtgtgtgagg
     4561 cgtctctctc ttcgtctctg ttgcgcaaac gcatagactg cactgaaaaa atcgattacc
     4621 tattttttat gaatgaatat ttgcactatt actattcaaa actattaaga tagcaatcac
     4681 attcaatagc caaatactat accacctgag cgatgcaacg aaatgatcaa tttgagcaaa
     4741 aatgctgcat atttaggacg gcatcattat agaaatgctt cttgctgtgt acttttctct
     4801 cgtctggcag ctgtttcgcc gttattgtta aaaccggctt aagttaggtg tgttttctac
     4861 gactagtgaa tgccctacta gaagatgtgt gttgcacaaa atgtccctgg aataaccaat
     4921 ttgaagtgca gatagcagta aacgtaagct aatatgaata ttatttaact gtaatgtttt
     4981 aatatcgctg gacattacta ataaacccac tataaacaca tgtacatatg tatgttttgg
     5041 catacaatga gtagttgggg aaaaaatgtg taaaagcacc gtgaccatca cagcataaag
     5101 ataaccagct gaagtatcga atatgagtaa cccccaaatt gaatcacatg ccgcaactga
     5161 taggacccat ggaagtacac tcttcatggc gatatacaag acacacacaa gcacgaacac
     5221 ccagttgcgg aggaaattct ccgtaaatga aaacccaatc ggcgaacaat tcatacccat
     5281 atatggtaaa agttttgaac gcgacttgag agcggagagc attgcggctg ataaggtttt
     5341 agcgctaagc gggctttata aaacgggctg cgggaccagt tttcatatca ctaccgtttg
     5401 agttcttgtg ctgtgtggat actcctcccg acacaaagcc gctccatcag ccagcagtcg
     5461 tctaatccag agaccccgga tccaggaatt cgcccttgtt taaacatggt gagcaagggc
     5521 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     5581 cacaagttca gcgtgagggg cgagggcgag ggcgatgcca ccaacggcaa gctgaccctg
     5641 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     5701 agccacggcg tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc
     5761 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     5821 acctacaaga cccgcgccga ggtgaagttc gagggcgaca ccctagtgaa ccgcatcgag
     5881 ctgaagggcg tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     5941 ttcaacagcc acaacatcta tatcatggcc gtcaagcaga agaacggcat caaggtgaac
     6001 ttcaagatcc gccacaacgt ggaggacggc agcgtgcagc tcgccgacca ctaccagcag
     6061 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca gccactacct gagcacccag
     6121 tccgtgctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttccgc
     6181 accgccgccg ggatcactct cggcatggac gagctgtaca agcttgaggg cagaggaagc
     6241 cttctaacat gcggtgacgt ggaggagaat cccggccctg ctagcggtag cggcagcggt
     6301 agcatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt cccccgggcc
     6361 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgacccg
     6421 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     6481 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     6541 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     6601 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     6661 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     6721 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     6781 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     6841 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     6901 tgactcgagg gatccagaca tgataagata cattgatgag tttggacaaa ccacaactag
     6961 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     7021 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     7081 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtatggc
     7141 tgattatgat cagtcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt
     7201 gggcgcgggg catgactatc gtcgccgcac ttatggtacg cgcccgggga gcccaagggc
     7261 acgccctggc acccgaaggg cgaattcact ggccgtcgtt ttacaacgtc gtgactggga
     7321 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     7381 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     7441 atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg
     7501 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     7561 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     7621 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     7681 ga
//
