LOCUS       sequence-354668-        7750 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for CRISPR-mediated knock-in line that expresses a QF2 
            transcriptional activator by knocking-in a T2A-QF2 fragment 
            replacing the stop codon of the endogenous Aedes aegypti Orco gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7750)
  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 7750)
  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"; 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: ORCO-T2A-QF2
FEATURES             Location/Qualifiers
     source          1..7750
                     /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             1624..1677
                     /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      3264..3273
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3270..3947
                     /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(3450..3470)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     3862..3885
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    4068..4189
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4105..4124)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4159..4178
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5529..5545)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5529..5545)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5542..5564)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5553..5569
                     /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(5577..5583,5584..5601,5602..5607))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5622..5643
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5760..5777)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5931..6519)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6011..6030)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6690..7481,7482..7550))
                     /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     7313..7332
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7551..7655)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7723..7741
                     /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 acccgggagt
      421 ttcgctttcc acgcgcggtg tgtctggaat cgttattaac gaaaaaaaat attctaattc
      481 gggatttaca attctctgtg aatatgaaaa catttcaacg agagaaacga aagttatagt
      541 gatttgaagg ttttcggcta ttctgtaaga tattctttca aataataggt gccaaattgg
      601 tggacatgtt ttgtaattaa cgatttcaga cacaccgtgc cacgtttggt tctctctagc
      661 aatttgattg tgcatttttt tttctctaac agagttcatc agtgatggag gctgcctact
      721 cgtgccactg gtatgacggt tccgaggaag ccaaaacttt cgtgcaaatc gtttgccagc
      781 agtgtcagaa agcgatgacc atatccgggg ccaagttttt caccgtttca ctggatctgt
      841 tcgcatcggt gagtgatttt tttttcgttt tcgattcggt ttctgggggg aaattagtgt
      901 cggatcgatc gataaaagcg attgcatggg cataaccaga aaggagacat gagtcctcct
      961 gcctccacct cccttttgaa aaaaagctgg gtgaattaat tttgtgtttg tattttcaaa
     1021 atcgtctata tctacaaaat aattgcatca cgattgatat ttctgagaaa tttttgaagt
     1081 tactcaaaca ataattttcc taaaaaagat cttatcagaa attatgagaa ggttttatca
     1141 acagtccgca tttttgaatc aatataataa gtttttctga aaaaaaaaaa atacagaaat
     1201 acggtgacga atttctgtgg aatttttttg agattcaaat atttcaaaaa catattttat
     1261 ggtcgtctgc aaaaaaccgt gagacgtttt tgatttggtg gaatctatct tgaagatttc
     1321 ttcgatgaac tgtatgaaat tactatgtcg aatcaattga atctgtagaa aaaatgtctc
     1381 atccggattt ttccaagcct tgtttctaat cgtccactgg actccacttt ggaaaagtgt
     1441 gaatttttta agtcacttgc ccctccggct attctggcta cgccaatgac cgattgtata
     1501 agaaagccac gaaactgagt caactataat taatgattcg tcgttttctt tccgttttcc
     1561 tttcccccac aggttctggg agcggtcgtc acctacttca tggtgctcgt gcagttgaaa
     1621 ggagagggca gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccctatg
     1681 ccacccaagc gcaaaacgct taacgctgcg gctgaggcta acgctcatgc cgacggacac
     1741 gccgacggaa acgccgacgg acacgtggcc aatacggccg cgtcctcgaa taatgcgagg
     1801 ttcgctgatc tcactaacat cgatactccg ggtctgggac ccacaactac gaccctgctc
     1861 gtggaaccag cacgctcaaa gcgtcaacga gtgtcccgcg catgcgacca gtgccgtgca
     1921 gcccgagaga aatgcgacgg aatacagcct gcgtgtttcc cgtgcgtttc ccagggaagg
     1981 tcctgcactt atcaggcttc gccgaaaaag aggggagttc aaaccggtta tattcgtacg
     2041 ctggagctcg ccctcgcctg gatgtttgaa aatgtcgcgc gttccgaaga tgccttgcat
     2101 aacctcctcg tccgtgacgc cggacaagga tcagctctgc tcgttggtaa agattcgccg
     2161 gctgccgagc gactccatgc ccgttgggct actagccgtg tcaataagag cattacccgc
     2221 ctcctccgtc agttggagct ccctcctacc gccacggcta cggcctcgat aatgccgcac
     2281 gtgatggagc agcctctcag taccagcatt aaccccgtca acgaccgctt caacggtatt
     2341 cccaacccca ctccgtataa ctccgatgca gctctcgatg ctatcactca gaccaacgat
     2401 tatggaagcg taaatacaca tggtatcctc tctacttacc cgccaccggc tacgcacctt
     2461 aatgaagctt ccgtcgctct cgctcccggt ggcgcccccc cccgaccgcc tcctccgtat
     2521 gttgacagca cgaccaatca cccgccgtac cactcgaatc tggttccaat ggcgaacttt
     2581 ggttactcga ccgttgatta cgatgccatg gttgacgatt tggctagcat tgaatacacg
     2641 gacgctgtgg atgtcgaccc acagtttatg accaatctgg gattcgttcc tggatgtaac
     2701 ttctccgaca ttaatacata cgaacagtga tgacgtctat cgataccgtc gactaaagcc
     2761 aaatagaaaa ttattcagtt cctggcttaa gtttttaaaa gtgatattat ttatttggtt
     2821 gtaaccaacc aaaagaatgt aaataactaa tacataatta tgttagtttt aagttagcaa
     2881 caaattgatt ttagctatat tagctacttg gttaataaat agaatatatt tatttaaaga
     2941 taattgcgtt tttattgtca gggagtgagt ttgcttaaaa actcgtttag atccaattcg
     3001 agctcgcccg gggatctaat tcaattagag actaattcaa ttagagctaa ttcaattagg
     3061 atccaagctt atcgatttcg aaccctcgac cgccggagta taaatagagg cgcttcgtct
     3121 acggagcgac aattcaattc aaacaagcaa agtgaacacg tcgctaagcg aaagctaagc
     3181 aaataaacaa gcgcagctga acaagctaaa caatcggggt accgctagag tcgacggtac
     3241 cgcgggcccg ggatccaccg gtcgccacca tggcctcctc cgagaacgtc atcaccgagt
     3301 tcatgcgctt caaggtgcgc atggagggca ccgtgaacgg ccacgagttc gagatcgagg
     3361 gcgagggcga gggccgcccc tacgagggcc acaacaccgt gaagctgaag gtgaccaagg
     3421 gcggccccct gcccttcgcc tgggacatcc tgtcccccca gttccagtac ggctccaagg
     3481 tgtacgtgaa gcaccccgcc gacatccccg actacaagaa gctgtccttc cccgagggct
     3541 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggcgaccgtg acccaggact
     3601 cctccctgca ggacggctgc ttcatctaca aggtgaagtt catcggcgtg aacttcccct
     3661 ccgacggccc cgtgatgcag aagaagacca tgggctggga ggcctccacc gagcgcctgt
     3721 acccccgcga cggcgtgctg aagggcgaga cccacaaggc cctgaagctg aaggacggcg
     3781 gccactacct ggtggagttc aagtccatct acatggccaa gaagcccgtg cagctgcccg
     3841 gctactacta cgtggacgcc aagctggaca tcacctccca caacgaggac tacaccatcg
     3901 tggagcagta cgagcgcacc gagggccgcc accacctgtt cctgtagcgg ccgcgactct
     3961 agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     4021 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     4081 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4141 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat aattcgttaa
     4201 ttatggacaa gctcaagact atccgatact atttttgata ctaatgcaac tgaacggcga
     4261 tacatgacat gaaatcgttt ttatatgtag atttataagg aagaaaaaaa ttatcagtac
     4321 aagaacaatt agactagacc ctccgacact gagtagcgga ggctaatgat gttgatatat
     4381 taaatatttt agtaagaatt tttgatgtag ctgaaataga cgctcaagat gaatgcaatg
     4441 aactctcgag aacttattca taacagtgag agaatgccct gattgttcac tgtaattagt
     4501 tttccacacg atttacttgg tttatgaaaa gcccttgtag attgagaagt cattagtcaa
     4561 gatatgtcac tcccgtgcaa aagtttgggt tcaccccctc aaacatatac aaaagtgttc
     4621 tgtccatatc tctgtgatta cacgtcaaat ttaaactctt taagccgcat tcgaaaggca
     4681 aagagttatt cttacttcgt atgtattttt ccaaaaacat ttttttaaat tttgtatact
     4741 aaatttttat taaagttgtg acatttttca aaaaacaccc ttaaaaatca tatctaattt
     4801 cctcagcatt gggttgacca aaatttcaaa tcaaagtgtc attagaatcg taatcttata
     4861 ttccttgaag agacaccacg aaatttcggc ggacaaatct ggaaagtatt caaaatcaat
     4921 gaaacagtca gtcaagtcat cgtgcaaaag tttgggttca tctctcagta tgatgcaaat
     4981 cgtgcaaaag tttgggttca cctgatccgc acgcacataa tttttgtcaa tatctcggcc
     5041 atttttcaac cgattttaat agtttaaagc ttttttgagc gcaaataatg gcgtgtacat
     5101 gattggtctg agatttcaca gatttaagta agttcaggtg aacccaaatt tttgcacgat
     5161 acaccataca ctgaggggtg aacccaaact tttgcacgat gacttgactg actgttttat
     5221 tgattttgaa tacttctcag atttttccgc caaaatttaa tgagtgctct tcaaagtata
     5281 taagattacg attctaatga caccttgttt taaaattttg gtcaattgaa tgctgaggaa
     5341 attagatatg atttttcagt gtgttttttg aaaaatgtcc caactttaag tacaaattta
     5401 gtatacaaag ttcaaagaat gtttttggaa aaatacatac gaagtaagaa taactctttg
     5461 cctttcgaat gcggcttaga gagttttcta gagtcgacct gcaggcatgc aagcttggcg
     5521 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     5581 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     5641 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5701 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     5761 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5821 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5881 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5941 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     6001 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     6061 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     6121 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     6181 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     6241 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6301 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     6361 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6421 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6481 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6541 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6601 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6661 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6721 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6781 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6841 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6901 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6961 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     7021 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     7081 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     7141 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     7201 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     7261 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     7321 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     7381 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     7441 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7501 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7561 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7621 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     7681 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     7741 ccctttcgtc
//
