LOCUS       sequence-354666-        8817 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Plasmid for CRISPR-mediated knock-in line that expresses the 
            activation domain of QF by knocking-in a T2A-QF-AD fragment 
            replacing the stop codon of the endogenous Aedes aegypti Ir25a gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8817)
  AUTHORS   Herre M, Goldman OV, Lu TC, Caballero-Vidal G, Qi Y, Gilbert ZN, 
            Gong Z, Morita T, Rahiel S, Ghaninia M, Ignell R, Matthews BJ, Li H,
            Vosshall LB, Younger MA
  TITLE     Non-canonical odor coding in the mosquito.
  JOURNAL   Cell. 2022 Aug 18;185(17):3104-3123.e28. doi: 
            10.1016/j.cell.2022.07.024.
  PUBMED    35985288
REFERENCE   2  (bases 1 to 8817)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cell.2022.07"; journalName: "Cell"; date: "2022-08-18- 
            18"; volume: "185"; issue: "17"; pages: "3104-3123.e28"
COMMENT     Sequence Label: IR25a-T2A-QF-AD
FEATURES             Location/Qualifiers
     source          1..8817
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2235..2288
                     /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             2319..2339
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          3123..3188
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             3318..3338
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3610..3731
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     regulatory      4000..4009
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4006..4008,4009..4011,4012..4725)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4051..4072)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4312..4331)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4659..4680
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4848..4969
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6596..6612)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6596..6612)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6609..6631)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6620..6636
                     /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)."
     promoter        complement(join(6644..6650,6651..6668,6669..6674))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6689..6710
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6827..6844)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6998..7586)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7078..7097)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7757..8548,8549..8617))
                     /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     8380..8399
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8618..8722)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8790..8808
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggatcg
      421 agtttatact gtggtggtaa gttattaata ttcttaagga tattccaggc ctttcaaaaa
      481 aacattgatt gattaccttt acctacaatc tttacattgt ttcgtagcaa gaaccattca
      541 ttatacgaga tccaactgct cccaaaggtt tcaaaggtta ttgtatcgac ttgttggatg
      601 aaattgcaaa aattgttaag ttcgattatg aaatcaagga agttgaagat ggcaagtttg
      661 gaaacatgaa cgaaaaaggt gaatggaacg gtatcgtacg caaattgatc gacaagcaag
      721 ccgatatcgg tttaggatcg atgtccgtga tggccgagcg ggaaactgta attgatttca
      781 cggtgccata ttacgatttg gttggaatat caatcatgat gctgctgccc agtacaccga
      841 gttcgctgtt caaatttttg accgtgttgg agacgaatgt gtggctttgt attctcgcag
      901 cgtatttctt cactagtttt ctcatgtgga tttttgatcg gtacagtcct tacagttacc
      961 agaacaatcg tgaaaagtac aagaatgatg atgaaaaacg agagttcaac attaaggaat
     1021 gcctatggtt ttgtatgact tcgttaacgc cgcaaggagg tggtgaagct ccgaaaaatt
     1081 tatccggacg gctggtggcg gcaacgtggt ggttgtttgg gtaagtattc tactgaattt
     1141 tgtttagata ataaatattt aatggatgat ttatgtaaaa cataaagatt catcatcatt
     1201 gcatcgtaca ctgctaactt ggctgctttc ctgaccgtat cgcgattgga cactcccgtg
     1261 gaatcgttgg acgatctttc gaagcagtat aaaattttgt acgccccttt gaacggatct
     1321 tcggctatga cttatttcca gcgaatggcg gatatcgaag cccgtttcta tgagtaagtt
     1381 tgttctcatt catgcttact aggggtaatt ttctaagcaa aatcactctc cacagaattt
     1441 ggaaagagat gtcgctgaat gattctctca cacccgtgga gcgctcgaag ctggcggttt
     1501 gggactatcc cgtcagtgat aagtatacga aaatgtggca agcaatgcag gaagctggac
     1561 tcccaaatag tttggatgaa gcggtcgagc gtattaggaa ctccacatcg gcctctggtt
     1621 tcgcttttct gggcgatgct actgatatac gatacaaagt acttacaaac tgtgatctac
     1681 agatggtcgg cgaagaattt tctcgcaaac cgtatgccat tgcagtacag caaggatcgc
     1741 cgttgaaaga tcaattcaac aatgcgtaag tttagctgac ggatgtccta ttcaatataa
     1801 caacttaata taccattata ccttctagca tcttgatgct tctcaacaaa cgacaacttg
     1861 aaaaactcaa ggaacaatgg tggaaaaatg atgacattca aagcaaatgt gaaaaaccgg
     1921 acgaccaatc cgatggtatt tcgattcaga acattggagg tgtcttcata gtgatcttcg
     1981 tcggcattgg aatggcgtgc attacgctgg tctttgaatt ctggtactac aaataccgca
     2041 aaaacgtaaa gattattgat gtcgccgagg ctacggaaga taaactggct cagaagacgt
     2101 ccaatttgag gttgccaaat ttgaagaacg aatttggagc catgggacag atcaaagatg
     2161 acagtcagaa aacgcaaagt ttgcgaactc gtacgcagac gatagacgcc aacaacttta
     2221 aatctcgttt tggagagggc agaggaagtc ttctaacatg cggtgacgtg gaggagaatc
     2281 ccggccctat ggataaagcg gaattaattc ccgagcctcc aaaaaagaag agaaaggtcg
     2341 aattggacgt ccgtcagttg gagctccctc ctaccgccac ggctacggcc tcgataatgc
     2401 cgcacgtgat ggagcagcct ctcagtacca gcattaaccc cgtcaacgac cgcttcaacg
     2461 gtattcccaa ccccactccg tataactccg atgcagctct cgatgctatc actcagacca
     2521 acgattatgg aagcgtaaat acacatggta tcctctctac ttacccgcca ccggctacgc
     2581 accttaatga agcttccgtc gctctcgctc ccggtggcgc ccccccccga ccgcctcctc
     2641 cgtatgttga cagcacgacc aatcacccgc cgtaccactc gaatctggtt ccaatggcga
     2701 actttggtta ctcgaccgtt gattacgatg ccatggttga cgatttggct agcattgaat
     2761 acacggacgc tgtggatgtc gacccacagt ttatgaccaa tctgggattc gttcctggat
     2821 gtaacttctc cgacattaat acatacgaac agagatctgg aggaggtggt ggaggtggag
     2881 gtggaggtac tagtctggag atcgaggccg ccttcctgga gcgcgagaac accgccctgg
     2941 agacccgcgt ggccgagctg cgccagcgcg tgcagcgcct gcgcaaccgc gtgagccagt
     3001 accgcacccg ctacggcccc ctgggcggcg gcaagtaagg ccggccgatc tttgtgaagg
     3061 aaccttactt ctgtggtgtg acataattgg acaaactacc tacagagatt taaagctcta
     3121 aggtaaatat aaaattttta agtgtataat gtgttaaact actgattcta attgtttgtg
     3181 tattttagat tccaacctat ggaactgatg aatgggagca gtggtggaat gcctttaatg
     3241 aggaaaacct gttttgctca gaagaaatgc catctagtga tgatgaggct actgctgact
     3301 ctcaacattc tactcctcca aaaaagaaga gaaaggtaga agaccccaag gactttcctt
     3361 cagaattgct aagttttttg agtcatgctg tgtttagtaa tagaactctt gcttgctttg
     3421 ctatttacac cacaaaggaa aaagctgcac tgctatacaa gaaaattatg gaaaaatatt
     3481 tgatgtatag tgccttgact agagatcata atcagccata ccacatttgt agaggtttta
     3541 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     3601 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     3661 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     3721 aatgtatctt atcatgtctg cgcccgggga tctaattcaa ttagagacta attcaattag
     3781 agctaattca attaggatcc aagcttatcg atttcgaacc ctcgaccgcc ggagtataaa
     3841 tagaggcgct tcgtctacgg agcgacaatt caattcaaac aagcaaagtg aacacgtcgc
     3901 taagcgaaag ctaagcaaat aaacaagcgc agctgaacaa gctaaacaat cggggtaccg
     3961 ctagagtcga cggtaccgcg ggcccgggat ccaccggtcg ccaccatggt gagcaagggc
     4021 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     4081 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     4141 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccttc
     4201 ggctacggcc tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc
     4261 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     4321 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     4381 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     4441 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     4501 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     4561 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagctaccag
     4621 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     4681 accgccgccg ggatcactct cggcatggac gagctgtaca agtaaagcgg ccgcgactct
     4741 agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     4801 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     4861 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4921 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa agcttatcga
     4981 tactgaagtt cattttttga gtagtaacgt gttgtttgaa attacccaat agagaagaaa
     5041 catatttttg tctatcgcat attaacaggt tccacttgaa tttttagatt tcttcaatta
     5101 tttttttttg tttcataatc agaagaaata tgaaaattgg actggatcag ttcttctttc
     5161 tcgactcgta tttttttctt ctcgaaaaaa ttggaacact atggaaaaac gcttaagatc
     5221 tctattctac cctagtttaa ctagtacaat aaacatcttg gctacattaa atttattctg
     5281 tggataaatc tgaacacaca ttttctacat cgatttatac taggattcag acccacagag
     5341 taatagaaat acaaccctgt ggaattcgca ttgtatcgta aagctagtgg gctacaatca
     5401 aagtgtagtc gtaaaaggtc tttttcgaaa atttttgaat tgtataaaag ttctcgcaaa
     5461 agatctaata tctagtatcc cacacaggat gcgatgcatg gaattccaac aacagcaaaa
     5521 aaaaacgcgt atcgaattta agatacgagg ggtagtaacg tgttgttata tcattcttga
     5581 tataatcgtt attttgagag tagtaacgtg ttagtaggat tggttatgta attttaatac
     5641 agtagctagt aagatcatcc caaatcattt aagggtgtat tatggaaatt gagtcgacct
     5701 catgagaaaa aagtcatctc aatttacatg attgtttcct tacaatgttt gcatccaatc
     5761 cgaaattcct taacaacttt tcatacattc tcgagcactt tatgcttaaa agccccgaag
     5821 gctttccggt ctgtctcgtt caacgtccac gtttcatgtc ggtaagaagc caccagatgt
     5881 atccgctttt cacgcatttt gtttttattt tggggttgtg gattttaagc tggttacaaa
     5941 gttctcccac cactaccaag cggttgcctg gttctctaga tttgtgctct tgaaaattcg
     6001 aggtttggct tcgatgcatc atcaccttaa tcttcatatg cgagataaac tggttgaacg
     6061 atcccccaaa atcgtagaga tcctagacta tagagaacaa tccaataatg tatgttaaag
     6121 ccgacgtcgg atagcgcttc caaaagaacc acgagtggga aaaaagaaag tgaagaaaat
     6181 ttgaaactgg cggtcaagaa atatccgaaa ccgaagacag tgtaacaaaa gaacttaggc
     6241 cgtcgttatt aattggagga agcggaatac gccaaaaccc tgaagtcgat gcgaagcact
     6301 gagaagctat ccgcctaatg acgacatcgt gcaaactagc atcgatgaaa tgaagttaaa
     6361 aaaacaaaca aactagtgta gtctgttgtg tggaagagcc ggaaggtggc ttggacgagg
     6421 tgctcaagaa ttggaggcca agatgctgag aacggggctg aaggtgcgga tgtcggttga
     6481 tcggacgacg agctcggaag tgctggagga actcggaagt caagaggggc ttttctatag
     6541 tagggtacaa aaatctagag tcgacctgca ggcatgcaag cttggcgtaa tcatggtcat
     6601 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     6661 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     6721 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     6781 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     6841 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     6901 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     6961 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     7021 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     7081 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     7141 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     7201 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     7261 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     7321 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     7381 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     7441 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     7501 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     7561 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     7621 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     7681 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     7741 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     7801 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     7861 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     7921 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     7981 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     8041 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     8101 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     8161 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     8221 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     8281 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     8341 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     8401 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     8461 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     8521 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     8581 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     8641 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     8701 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     8761 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtc
//
