LOCUS       sequence-257080-        7212 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 ppk301 gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7212)
  AUTHORS   Matthews BJ, Younger MA, Vosshall LB
  TITLE     The ion channel ppk301 controls freshwater egg-laying in the 
            mosquito Aedes aegypti.
  JOURNAL   eLife. 2019 May 21;8. pii: 43963. doi: 10.7554/eLife.43963.
  PUBMED    31112133
REFERENCE   2  (bases 1 to 7212)
  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: "2019-05-21"; volume: "8. pii: 43963. doi"; pages: " 
            10.7554/eLife.43963"
COMMENT     Sequence Label: ppk301-T2A-QF2 HDR plasmid
FEATURES             Location/Qualifiers
     source          1..7212
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1..23)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     123..142
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     336..358
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     350..367
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     351..367
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1378..1431
                     /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      3018..3027
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3024..3701
                     /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(3204..3224)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     3616..3639
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3822..3943
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3859..3878)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3913..3932
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4963..4979)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4963..4979)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4976..4998)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4987..5003
                     /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(5011..5017,5018..5035,5036..5041))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5056..5077
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5194..5211)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5365..5953)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5445..5464)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6124..6915,6916..6984))
                     /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     6747..6766
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6985..7089)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7157..7175
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
       61 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
      121 tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag
      181 atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg
      241 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
      301 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
      361 ggccagtgaa ttctgtgttc gtcggatttc gcagccaatt ctttgactac cgttttatat
      421 tcgtggtgaa ttcaattatt tttatggctt ttatattatc ataaaatcga acaatattag
      481 ttgagagata tacagaaaat cgattgcgat ttctttgata aatttaaaag atatggcata
      541 cacattttat aaaataaaca tccctgtacg ttgatttttt attttcctta cagtcccatt
      601 tgctacaaca atgagatctt caaacgagcg tctatttcgc agatcatttc acggagcaca
      661 atccaagttg atacaatttg acgaaagtaa atatcatgcc gcccttatga gtctcgcttt
      721 gaataaaata tctaccttcc aataggcaaa gcaccgaaag ttacattcgg cgagaatttc
      781 cgagagtatt gcctgaatac gactatccac ggcttgaagt acatcggaac gataagtctc
      841 tctgcgatag aaaggttttc aaaatagcaa aatgacccaa aacgtgatca aatattcaac
      901 cttatttctc acaggtgctt ctttttcatc tcgttcatcc ttgtctcaat tctttcgatt
      961 tacttcatca ccaacgtgta tcaaaagtgg caatcttcgc cgatcataat tggtctcagt
     1021 ccgaaggcaa cccacatcag agacatccct tttccggcgg ttaccatttg caatatgaat
     1081 caagccactc gaacggctgc ggaagcaata aagcctaaca ctctcgagaa agcgattctg
     1141 aacagtatct gttcgatgga tggcgagttc aatatgacca attacgaggg taaatggtcc
     1201 actgtgaaaa aaatgctact ctctgctact caaccgtgtc acagtatggt ggaagcttgt
     1261 cggtatgcgc agaatatcta caactgtaga cacatgttcc gccctgtgtt gaccgatgaa
     1321 ggcctatgct gtacattcaa cagcgttgac ccgtcgtatc tcctatggaa ccggggagag
     1381 ggcagaggaa gtcttctaac atgcggtgac gtggaggaga atcccggccc tatgccaccc
     1441 aagcgcaaaa cgcttaacgc tgcggctgag gctaacgctc atgccgacgg acacgccgac
     1501 ggaaacgccg acggacacgt ggccaatacg gccgcgtcct cgaataatgc gaggttcgct
     1561 gatctcacta acatcgatac tccgggtctg ggacccacaa ctacgaccct gctcgtggaa
     1621 ccagcacgct caaagcgtca acgagtgtcc cgcgcatgcg accagtgccg tgcagcccga
     1681 gagaaatgcg acggaataca gcctgcgtgt ttcccgtgcg tttcccaggg aaggtcctgc
     1741 acttatcagg cttcgccgaa aaagagggga gttcaaaccg gttatattcg tacgctggag
     1801 ctcgccctcg cctggatgtt tgaaaatgtc gcgcgttccg aagatgcctt gcataacctc
     1861 ctcgtccgtg acgccggaca aggatcagct ctgctcgttg gtaaagattc gccggctgcc
     1921 gagcgactcc atgcccgttg ggctactagc cgtgtcaata agagcattac ccgcctcctc
     1981 cgtcagttgg agctccctcc taccgccacg gctacggcct cgataatgcc gcacgtgatg
     2041 gagcagcctc tcagtaccag cattaacccc gtcaacgacc gcttcaacgg tattcccaac
     2101 cccactccgt ataactccga tgcagctctc gatgctatca ctcagaccaa cgattatgga
     2161 agcgtaaata cacatggtat cctctctact tacccgccac cggctacgca ccttaatgaa
     2221 gcttccgtcg ctctcgctcc cggtggcgcc cccccccgac cgcctcctcc gtatgttgac
     2281 agcacgacca atcacccgcc gtaccactcg aatctggttc caatggcgaa ctttggttac
     2341 tcgaccgttg attacgatgc catggttgac gatttggcta gcattgaata cacggacgct
     2401 gtggatgtcg acccacagtt tatgaccaat ctgggattcg ttcctggatg taacttctcc
     2461 gacattaata catacgaaca gtgatgacgt ctatcgatac cgtcgactaa agccaaatag
     2521 aaaattattc agttcctggc ttaagttttt aaaagtgata ttatttattt ggttgtaacc
     2581 aaccaaaaga atgtaaataa ctaatacata attatgttag ttttaagtta gcaacaaatt
     2641 gattttagct atattagcta cttggttaat aaatagaata tatttattta aagataattg
     2701 cgtttttatt gtcagggagt gagtttgctt aaaaactcgt ttagatccaa ttcgagctcg
     2761 cccggggatc taattcaatt agagactaat tcaattagag ctaattcaat taggatccaa
     2821 gcttatcgat ttcgaaccct cgaccgccgg agtataaata gaggcgcttc gtctacggag
     2881 cgacaattca attcaaacaa gcaaagtgaa cacgtcgcta agcgaaagct aagcaaataa
     2941 acaagcgcag ctgaacaagc taaacaatcg gggtaccgct agagtcgacg gtaccgcggg
     3001 cccgggatcc accggtcgcc accatggcct cctccgagaa cgtcatcacc gagttcatgc
     3061 gcttcaaggt gcgcatggag ggcaccgtga acggccacga gttcgagatc gagggcgagg
     3121 gcgagggccg cccctacgag ggccacaaca ccgtgaagct gaaggtgacc aagggcggcc
     3181 ccctgccctt cgcctgggac atcctgtccc cccagttcca gtacggctcc aaggtgtacg
     3241 tgaagcaccc cgccgacatc cccgactaca agaagctgtc cttccccgag ggcttcaagt
     3301 gggagcgcgt gatgaacttc gaggacggcg gcgtggcgac cgtgacccag gactcctccc
     3361 tgcaggacgg ctgcttcatc tacaaggtga agttcatcgg cgtgaacttc ccctccgacg
     3421 gccccgtgat gcagaagaag accatgggct gggaggcctc caccgagcgc ctgtaccccc
     3481 gcgacggcgt gctgaagggc gagacccaca aggccctgaa gctgaaggac ggcggccact
     3541 acctggtgga gttcaagtcc atctacatgg ccaagaagcc cgtgcagctg cccggctact
     3601 actacgtgga cgccaagctg gacatcacct cccacaacga ggactacacc atcgtggagc
     3661 agtacgagcg caccgagggc cgccaccacc tgttcctgta gcggccgcga ctctagatca
     3721 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     3781 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     3841 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     3901 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttactcaact gccaaccgta
     3961 cccttgtgcc cgataatcct tttgtaccga ttgaatggac gccagaaaca ggatttatcg
     4021 gagaagttac caactcgacc ttcccgaggt acatcgctgg gactggggct tccatgggtc
     4081 tcacggtcgt tctagatgcc aacgtcaagg actactactg ttcctcaaca agttcgtacg
     4141 gtttcaaact gatcctgcac aaccccacgg aaactccgaa gatggccgac tatgctcact
     4201 acatccaggt tgggacggag aataggatcg tcgtaactcc gaagcttagc gatgcatcct
     4261 acctgatccg taagacctcg caagagacga gacaatgcgt ttttgccagt gaggccaact
     4321 tgtcgtactt tcgaacttac tcacgaaaca actgcgaaat ggaatgcgag gcccgtttga
     4381 ttcaggacaa ctgcggttgc gtattgtact acatgccgaa gctggaggag gacaacaaaa
     4441 tctgcagcaa agccgatgca ggatgttacg agaagatccg gtcgtcgata gctcaaacgg
     4501 cgaacaatac gctgtcgtgc agttgtctac cgggttgttt cgagatcagt tactcgatcg
     4561 atcgatcttc ggctgatctc tgcgtgggga agttcagagt tcgcgagaac ttgctggacg
     4621 ttaacgattc gtatgcaagg tatgtatact tctgatctac agaaaaccct tcgagatgaa
     4681 tgcagttaat caatttcagg gaaaacatcg ctctggtcta catcttcttt tccgagacat
     4741 acttcagaag cttctccaaa ggagagctga ttgggttcac cgaattcctg tcgaacgttg
     4801 gaggtttgct tggtttgttc atgggttttt cgttgatctc tctggctgaa gtgatctact
     4861 tcataaccct tcgcccactg ttcgccaaac gaaaggaaaa gcaggatcgc gccaaacaag
     4921 ataatttcat acgggatcga cactaagctt ggcgtaatca tggtcatagc tgtttcctgt
     4981 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5041 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5101 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5161 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     5221 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5281 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5341 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5401 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5461 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5521 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5581 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5641 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5701 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5761 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     5821 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5881 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5941 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     6001 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     6061 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     6121 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6181 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     6241 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     6301 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6361 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6421 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6481 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6541 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6601 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6661 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6721 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     6781 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6841 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6901 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6961 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     7021 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     7081 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     7141 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga
     7201 tgacggtgaa aa
//
