LOCUS       sequence-274103-        7461 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 7461)
  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 7461)
  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..7461
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(146..469)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     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"
     misc_feature    396..452
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(465..481)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(465..481)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(478..500)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    489..505
                     /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(513..519,520..537,538..543))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    558..579
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(696..713)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(867..1455)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(947..966)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1626..2417,2418..2486))
                     /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     2249..2268
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2487..2591)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2659..2677
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             3484..3537
                     /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      5124..5133
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5130..5807
                     /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(5310..5330)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     5722..5745
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    5928..6049
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5965..5984)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6019..6038
                     /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 cctctagagt cgacctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct
      481 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
      541 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
      601 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
      661 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
      721 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
      781 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
      841 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
      901 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
      961 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     1021 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     1081 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     1141 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     1201 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     1261 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     1321 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     1381 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     1441 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     1501 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     1561 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     1621 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     1681 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     1741 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     1801 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     1861 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     1921 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     1981 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     2041 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     2101 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     2161 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     2221 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     2281 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     2341 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     2401 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     2461 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     2521 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     2581 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     2641 atgacattaa cctataaaaa taggcgtatc acgaggcctc gctcattatt attggtaccg
     2701 gaatggaggc tatccattcc aatgatggac cggaataaat cttcttttcc cagcagcgca
     2761 tttgcatcgc cgtatactat tttcacatct tgttttgggg actctccata taccttatct
     2821 agactctcat agaactcatc cttcatgtca tccggcttat cgttctttgg cacatatata
     2881 gtgaacattt tgtagatgaa gaacttgtcc ttcatttaca acacacagat tcggttgctt
     2941 aacggtttcc atcaaataac tcgcgtcatc tgcttcgcaa tcactatgaa gccaactcca
     3001 cgttggtatt gctctgttgc cgccgctgta gtagatgtgg tacttgaatg aagtgttggc
     3061 gatgcaccgt ttcgtgttcg tagaaagcca ccggaagtat gattaactga taacttaatt
     3121 acttgcatat ctgaaaatga taaaaatcat atggaaatgt cggaaaaata aaaacaatta
     3181 gtactttcca tggattactg agcctaattg aatatctaga tataatttat atggaaaaca
     3241 aactctcgat gaactcaaac atatgttgac cataactttg tagcacctaa ttgatttatt
     3301 ttccatttcc tctccttttg tttcagctcc ttcaagttac tgttgcaagt tctcaatagt
     3361 atgtcatcgc cgcctgcaat gcccaattac aataaccgtt acccggcatt gaacggctat
     3421 ccccagatca acggtgtcga cccggcagaa cccatcgatt gtcgtaagct tcaatcgcac
     3481 ggagagggca gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccctatg
     3541 ccacccaagc gcaaaacgct taacgctgcg gctgaggcta acgctcatgc cgacggacac
     3601 gccgacggaa acgccgacgg acacgtggcc aatacggccg cgtcctcgaa taatgcgagg
     3661 ttcgctgatc tcactaacat cgatactccg ggtctgggac ccacaactac gaccctgctc
     3721 gtggaaccag cacgctcaaa gcgtcaacga gtgtcccgcg catgcgacca gtgccgtgca
     3781 gcccgagaga aatgcgacgg aatacagcct gcgtgtttcc cgtgcgtttc ccagggaagg
     3841 tcctgcactt atcaggcttc gccgaaaaag aggggagttc aaaccggtta tattcgtacg
     3901 ctggagctcg ccctcgcctg gatgtttgaa aatgtcgcgc gttccgaaga tgccttgcat
     3961 aacctcctcg tccgtgacgc cggacaagga tcagctctgc tcgttggtaa agattcgccg
     4021 gctgccgagc gactccatgc ccgttgggct actagccgtg tcaataagag cattacccgc
     4081 ctcctccgtc agttggagct ccctcctacc gccacggcta cggcctcgat aatgccgcac
     4141 gtgatggagc agcctctcag taccagcatt aaccccgtca acgaccgctt caacggtatt
     4201 cccaacccca ctccgtataa ctccgatgca gctctcgatg ctatcactca gaccaacgat
     4261 tatggaagcg taaatacaca tggtatcctc tctacttacc cgccaccggc tacgcacctt
     4321 aatgaagctt ccgtcgctct cgctcccggt ggcgcccccc cccgaccgcc tcctccgtat
     4381 gttgacagca cgaccaatca cccgccgtac cactcgaatc tggttccaat ggcgaacttt
     4441 ggttactcga ccgttgatta cgatgccatg gttgacgatt tggctagcat tgaatacacg
     4501 gacgctgtgg atgtcgaccc acagtttatg accaatctgg gattcgttcc tggatgtaac
     4561 ttctccgaca ttaatacata cgaacagtga tgacgtctat cgataccgtc gactaaagcc
     4621 aaatagaaaa ttattcagtt cctggcttaa gtttttaaaa gtgatattat ttatttggtt
     4681 gtaaccaacc aaaagaatgt aaataactaa tacataatta tgttagtttt aagttagcaa
     4741 caaattgatt ttagctatat tagctacttg gttaataaat agaatatatt tatttaaaga
     4801 taattgcgtt tttattgtca gggagtgagt ttgcttaaaa actcgtttag atccaattcg
     4861 agctcgcccg gggatctaat tcaattagag actaattcaa ttagagctaa ttcaattagg
     4921 atccaagctt atcgatttcg aaccctcgac cgccggagta taaatagagg cgcttcgtct
     4981 acggagcgac aattcaattc aaacaagcaa agtgaacacg tcgctaagcg aaagctaagc
     5041 aaataaacaa gcgcagctga acaagctaaa caatcggggt accgctagag tcgacggtac
     5101 cgcgggcccg ggatccaccg gtcgccacca tggcctcctc cgagaacgtc atcaccgagt
     5161 tcatgcgctt caaggtgcgc atggagggca ccgtgaacgg ccacgagttc gagatcgagg
     5221 gcgagggcga gggccgcccc tacgagggcc acaacaccgt gaagctgaag gtgaccaagg
     5281 gcggccccct gcccttcgcc tgggacatcc tgtcccccca gttccagtac ggctccaagg
     5341 tgtacgtgaa gcaccccgcc gacatccccg actacaagaa gctgtccttc cccgagggct
     5401 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggcgaccgtg acccaggact
     5461 cctccctgca ggacggctgc ttcatctaca aggtgaagtt catcggcgtg aacttcccct
     5521 ccgacggccc cgtgatgcag aagaagacca tgggctggga ggcctccacc gagcgcctgt
     5581 acccccgcga cggcgtgctg aagggcgaga cccacaaggc cctgaagctg aaggacggcg
     5641 gccactacct ggtggagttc aagtccatct acatggccaa gaagcccgtg cagctgcccg
     5701 gctactacta cgtggacgcc aagctggaca tcacctccca caacgaggac tacaccatcg
     5761 tggagcagta cgagcgcacc gagggccgcc accacctgtt cctgtagcgg ccgcgactct
     5821 agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     5881 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     5941 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     6001 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttag agcggggttt
     6061 ccatagacac aaaattaagg cgagaaatcg ccaaccgtat aaccattgct gatcttacca
     6121 gcacacatca tcatggaatc ttctgaccga tcgcgtcatc gatcgtgtct tgtacattta
     6181 ccatgcgagc ttgaatcaag ggaaacacac atatgtacta taaaaaaata aagaaatgat
     6241 aaatcttgca atgaatatac cttatctaat aaatataaca atcaaaaaaa cgccagccgt
     6301 ctatcaaaga tgagaaatca aatacacctg caaacatttg atatcctgta aaaaaaacga
     6361 aaagcagtac taatagacaa atacgataat aatatacgat aataacgtca aacgtagcag
     6421 caattcaggt taaccatttt atcaaagtcg ttatatttaa acttttaaaa gtagttgaag
     6481 tgagaaataa aaagcatatc tttaaccgtt cctcctggag aaagtgtaca aggaaaagta
     6541 tacaattgtg aactgtaaaa aaagttgtaa caaacgagct gataaatatt gcttaaattt
     6601 tcgaaatata catttcaaaa agactaacta taagtgcaca gaaactttct tcgcttcagt
     6661 agtgtttcgt cgaacaatac gcggtagtca tgctttgctt cctgtagtga tattcgccag
     6721 tttatcatca taacaaaaag ttaacccagt taaagcaaaa aagaacatta acaagacatg
     6781 taaatgatcc gtagagtttc cgagtacaaa acgaacttag ctaaacaaaa gaaaaacgaa
     6841 tgaaatgcaa ccagtgaatc atatagcatc atttaaccca atttattacc gatataaacc
     6901 tagttacgta atttgtacat taaaacaaaa gagttaaacc aaactattta accctcttcc
     6961 aacaatttaa acaaaaaaaa aaactccccg cgtacaattg atcgattcaa gcatctaaac
     7021 tcccaccgaa ctaaaaaaaa tgtgacgttt cccttgcaac gaacaaccat ccgaaactgg
     7081 caacgcttct gtgttcgtca acgaacgcca catccgaaaa atctgcctgt cagatcatca
     7141 atctactgtt ctatccgaaa tctaccggcg tttcacgccg catctacaag cgcacctgtc
     7201 aacatccgag atacgaagca cgggcgcaaa gcgtttgcca gcagttctcc ctcaatcagc
     7261 gaatcaacac gtaaatctgg gccactgtac tgtcagcggc cgcagtaacc atgggacggt
     7321 ttagccgacc tttaacggct ggtaagtact ttggtgggag tcactcacga cgtttttttt
     7381 gggtgactaa actccggtag acaatcatgg tggtctcctt ggtgaatgca aatcggaata
     7441 atggcttcgc cactgtttgc c
//
