LOCUS Exported 7212 bp ds-DNA circular SYN 12-MAY-2021 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 ppk301-T2A-QF2 HDR plasmid 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 May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7212 /organism="synthetic DNA construct" /mol_type="other DNA" 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 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" 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(5011..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(6124..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 //