LOCUS       sequence-292289-        7488 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for CRISPR-generated knock-in line that expresses a QF2 
            transcriptional activator by knocking-in a T2A-QF2 fragment into the
            coding sequence of the endogenous Aedes aegypti fru gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7488)
  AUTHORS   Basrur NS, De Obaldia ME, Morita T, Herre M, von Heynitz RK, 
            Tsitohay YN, Vosshall LB
  TITLE     Fruitless mutant male mosquitoes gain attraction to human odor.
  JOURNAL   Elife. 2020 Dec 7;9. pii: 63982. doi: 10.7554/eLife.63982.
  PUBMED    33284111
REFERENCE   2  (bases 1 to 7488)
  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: "Elife."; 
            date: "2020-12-7"; volume: "9. pii: 63982. doi"; pages: " 
            10.7554/eLife.63982"
COMMENT     Sequence Label: fru-T2A-QF2 HDR plasmid
FEATURES             Location/Qualifiers
     source          1..7488
                     /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"
     primer_bind     complement(487..503)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(487..503)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(500..522)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    511..527
                     /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(535..541,542..559,560..565))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    580..601
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(718..735)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(889..1477)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(969..988)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1648..2439,2440..2508))
                     /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     2271..2290
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2509..2613)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2681..2699
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             3506..3559
                     /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"
     regulatory      5146..5155
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5152..5829
                     /codon_start=1
                     /product="improved tetrameric variant of DsRed fluorescent 
                     protein"
                     /label=DsRed2
                     /note="mammalian codon-optimized"
                     /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     primer_bind     complement(5332..5352)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     5744..5767
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    5950..6071
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5987..6006)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6041..6060
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse 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 acccggggat
      421 ccgcgagggc gatgccacct acggcctaga gtcgacctgc aggcatgcaa gcttggcgta
      481 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
      541 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
      601 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
      661 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
      721 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
      781 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
      841 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
      901 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
      961 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     1021 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     1081 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     1141 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     1201 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     1261 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     1321 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     1381 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     1441 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     1501 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     1561 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     1621 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     1681 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     1741 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     1801 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     1861 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     1921 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     1981 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     2041 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     2101 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     2161 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     2221 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     2281 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     2341 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     2401 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     2461 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     2521 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     2581 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     2641 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     2701 tcgctcatta ttattggtac cggaatggag gctatccatt ccaatgatgg accggaataa
     2761 atcttctttt cccagcagcg catttgcatc gccgtatact attttcacat cttgttttgg
     2821 ggactctcca tataccttat ctagactctc atagaactca tccttcatgt catccggctt
     2881 atcgttcttt ggcacatata tagtgaacat tttgtagatg aagaacttgt ccttcattta
     2941 caacacacag attcggttgc ttaacggttt ccatcaaata actcgcgtca tctgcttcgc
     3001 aatcactatg aagccaactc cacgttggta ttgctctgtt gccgccgctg tagtagatgt
     3061 ggtacttgaa tgaagtgttg gcgatgcacc gtttcgtgtt cgtagaaagc caccggaagt
     3121 atgattaact gataatttaa ttacttgcat atctgaaaat gataaaaatc atatggaaat
     3181 gtcggaaaaa taaaaacaat cagtactttc catggattac tgagcctaat tgaatatcta
     3241 gatataattt atatggaaaa caaactctcg atgaactcaa acatatgttg accataactt
     3301 tgtagcacct aattgattta ttttccattt cctctccttt tgtttcagct ccttcaagtt
     3361 actgttgcaa gttctcaata gtatgtcatc gccgcctgca atgcccaatt acaataaccg
     3421 ttacccggca ttgaacggct atccccagat caacggtgtc gacccggcag aacccatcga
     3481 ttgtcgtaag cttcaatcgc acggagaggg cagaggaagt cttctaacat gcggtgacgt
     3541 ggaggagaat cccggcccta tgccacccaa gcgcaaaacg cttaacgctg cggctgaggc
     3601 taacgctcat gccgacggac acgccgacgg aaacgccgac ggacacgtgg ccaatacggc
     3661 cgcgtcctcg aataatgcga ggttcgctga tctcactaac atcgatactc cgggtctggg
     3721 acccacaact acgaccctgc tcgtggaacc agcacgctca aagcgtcaac gagtgtcccg
     3781 cgcatgcgac cagtgccgtg cagcccgaga gaaatgcgac ggaatacagc ctgcgtgttt
     3841 cccgtgcgtt tcccagggaa ggtcctgcac ttatcaggct tcgccgaaaa agaggggagt
     3901 tcaaaccggt tatattcgta cgctggagct cgccctcgcc tggatgtttg aaaatgtcgc
     3961 gcgttccgaa gatgccttgc ataacctcct cgtccgtgac gccggacaag gatcagctct
     4021 gctcgttggt aaagattcgc cggctgccga gcgactccat gcccgttggg ctactagccg
     4081 tgtcaataag agcattaccc gcctcctccg tcagttggag ctccctccta ccgccacggc
     4141 tacggcctcg ataatgccgc acgtgatgga gcagcctctc agtaccagca ttaaccccgt
     4201 caacgaccgc ttcaacggta ttcccaaccc cactccgtat aactccgatg cagctctcga
     4261 tgctatcact cagaccaacg attatggaag cgtaaataca catggtatcc tctctactta
     4321 cccgccaccg gctacgcacc ttaatgaagc ttccgtcgct ctcgctcccg gtggcgcccc
     4381 cccccgaccg cctcctccgt atgttgacag cacgaccaat cacccgccgt accactcgaa
     4441 tctggttcca atggcgaact ttggttactc gaccgttgat tacgatgcca tggttgacga
     4501 tttggctagc attgaataca cggacgctgt ggatgtcgac ccacagttta tgaccaatct
     4561 gggattcgtt cctggatgta acttctccga cattaataca tacgaacagt gatgacgtct
     4621 atcgataccg tcgactaaag ccaaatagaa aattattcag ttcctggctt aagtttttaa
     4681 aagtgatatt atttatttgg ttgtaaccaa ccaaaagaat gtaaataact aatacataat
     4741 tatgttagtt ttaagttagc aacaaattga ttttagctat attagctact tggttaataa
     4801 atagaatata tttatttaaa gataattgcg tttttattgt cagggagtga gtttgcttaa
     4861 aaactcgttt agatccaatt cgagctcgcc cggggatcta attcaattag agactaattc
     4921 aattagagct aattcaatta ggatccaagc ttatcgattt cgaaccctcg accgccggag
     4981 tataaataga ggcgcttcgt ctacggagcg acaattcaat tcaaacaagc aaagtgaaca
     5041 cgtcgctaag cgaaagctaa gcaaataaac aagcgcagct gaacaagcta aacaatcggg
     5101 gtaccgctag agtcgacggt accgcgggcc cgggatccac cggtcgccac catggcctcc
     5161 tccgagaacg tcatcaccga gttcatgcgc ttcaaggtgc gcatggaggg caccgtgaac
     5221 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg ccacaacacc
     5281 gtgaagctga aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtccccc
     5341 cagttccagt acggctccaa ggtgtacgtg aagcaccccg ccgacatccc cgactacaag
     5401 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
     5461 gtggcgaccg tgacccagga ctcctccctg caggacggct gcttcatcta caaggtgaag
     5521 ttcatcggcg tgaacttccc ctccgacggc cccgtgatgc agaagaagac catgggctgg
     5581 gaggcctcca ccgagcgcct gtacccccgc gacggcgtgc tgaagggcga gacccacaag
     5641 gccctgaagc tgaaggacgg cggccactac ctggtggagt tcaagtccat ctacatggcc
     5701 aagaagcccg tgcagctgcc cggctactac tacgtggacg ccaagctgga catcacctcc
     5761 cacaacgagg actacaccat cgtggagcag tacgagcgca ccgagggccg ccaccacctg
     5821 ttcctgtagc ggccgcgact ctagatcata atcagccata ccacatttgt agaggtttta
     5881 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     5941 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     6001 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     6061 aatgtatctt agagcggggt ttccatagac acaaaattaa ggcgagaaat cgccaaccgt
     6121 ataaccattg ctgatcttac cagcacacat catcatggaa tcttctgacc gatcgcgtca
     6181 tcgatcgtgt cttgtacatt taccatgcga gcttgaatca agggaaacac acatatgtac
     6241 tataaaaaaa taaagaaatg ataaatcttg caatgaatat accttatcta ataaatataa
     6301 caatcaaaaa aacgccagcc gtctatcaaa tatgagaaat caaatacacc tgcaaacatt
     6361 tgatatcctg taaaaaaaac gaaaagcagt actaatagac aaatacgata ataatatacg
     6421 ataataacgt caaacgtagc agcaattcag gttaaccatt ttatcaaaac cgttatattt
     6481 aaacttttaa aagtagttga agtgagaaat aaaaagcata tctttaaccg ttcctcctgg
     6541 agaaagtgta caaggaaaag tatacaattg tgaactgtaa aaaaagttgt aacaaacgag
     6601 ctgataaata ttgcttaaat tttcgaaata tacatttcaa aaagactaac tataagtgca
     6661 cagaaacttt cttcgcttca gtagtgtttc gtcgaacaat acgcggtagt catgctttgc
     6721 ttcctgtagt gatattcgcc agtttatcat cataacaaaa agttaaccca gttaaagcaa
     6781 aaaagaacat taacaagaca tgtaaatgat ccgtagagtt tccgagtaca aaacgaactt
     6841 agctaaacaa aagaaaaacg aatgaattgc aaccagtgaa tcatatagca tcatttaacc
     6901 caatttatta ccgatataaa cctagttacg taatttgtac attaaaacaa aagagttaaa
     6961 ccaaactatt taaccctctt ccaacaattt aaacaaaaaa aaaactcccc gcgtacaatt
     7021 gatcgattca agcatctaaa ctctcaccaa actaaaaaaa aatgtgacgt ttcccttgca
     7081 acgaacaacc atccgaaact ggcaacgctt ccgtgttcgt caacgaacgc cacatccgaa
     7141 aaatctgcct gtcagatcat caatctactg ttctatccga aatctaccgg cgtttcacgc
     7201 cgcatctact agtgcacctg tcaacatccg agatacgaag cacgggcgca aagcgtttgc
     7261 cagcacttct ccctcaatca gcaaatcatc acgtaaatct gggccactgt actgtcagcg
     7321 gccgcagtaa ccatggggcg gtttagccga cctttaacgg ctggtaagta ctttggtggg
     7381 agtcactcac gacggttttt tttttttggg taactaaact ccggtagaca atcatggtgg
     7441 tctccttggt gaatgcaaat cggaaaaatg gcttcgccac tgtttgcc
//
