LOCUS       sequence-362582-        8179 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HACK-compatible plasmid for generating T2A-QF2 knockins in Anopheles
            mosquitoes with a mCherry selectable marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8179)
  AUTHORS   Raji JI, Konopka JK, Potter CJ
  TITLE     A spatial map of antennal-expressed ionotropic receptors in the 
            malaria mosquito.
  JOURNAL   Cell Rep. 2023 Feb 10;42(2):112101. doi: 
            10.1016/j.celrep.2023.112101.
  PUBMED    36773296
REFERENCE   2  (bases 1 to 8179)
  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: "Cell Rep.";
            date: "2023-02-10"; volume: "42(2):112101. doi"; pages: " 
            10.1016/j.celrep.2023.112101"
COMMENT     Sequence Label: pHACK-QF2-AgU6-mCherry
FEATURES             Location/Qualifiers
     source          1..8179
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(129..197,198..989)
                     /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     complement(347..366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1160..1748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1649..1668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1902..1919
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2036..2057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2072..2077,2078..2095,2096..2102)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2110..2126
                     /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)."
     primer_bind     2115..2137
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2134..2150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2134..2150
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   2641..2675
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     misc_RNA        complement(2789..2864)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        complement(2987..3062)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    3205..3238
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3416..4126
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3561..3579)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    4246..4367
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    4431..4464
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(5797..5850)
                     /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"
     polyA_signal    5922..6043
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6167..6880)
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(6207..6227)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     6822..6850
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     repeat_region   7372..7434
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(7564..7581)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7564..7580)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7573..7595)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7722..8177
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7809..7828)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8019..8040
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
       61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
      121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
      181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
      241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
      301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     1861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     1921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     1981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     2041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     2101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     2161 ccaagcgcgc ccgccgggta actcacgggg tatccatgtc catttctgcg gcatccagcc
     2221 aggatacccg tcctcgctga cgtaatatcc cagcgccgca ccgctgtcat taatctgcac
     2281 accggcacgg cagttccggc tgtcgccggt attgttcggg ttgctgatgc gcttcgggct
     2341 gaccatccgg aactgtgtcc ggaaaagccg cgacgaactg gtatcccagg tggcctgaac
     2401 gaacagttca ccgttaaagg cgtgcatggc cacaccttcc cgaatcatca tggtaaacgt
     2461 gcgttttcgc tcaacgtcaa tgcagcagca gtcatcctcg gcaaactctt tccatgccgc
     2521 ttcaacctcg cgggaaaagg cacgggcttc ttcctccccg atgcccagat agcgccagct
     2581 tgggcgatga ctgagccgga aaaaagaccc gacgatatga tcctgatgca gctagattaa
     2641 ccctagaaag atagtctgcg taaaattgac gcatgcattc ttgaaatatt gctctctctt
     2701 tctaaatagc gcgaatccgt cgctgtgcat ttaggacatc tcagtcttaa ttaattgaac
     2761 aactctcagg ctccaggtag gcaaaaaagc accgactcgg tgccactttt tcaagttgat
     2821 aacggactag ccttatttta acttgctatt tctagctcta aaacaggtct tctcgaagac
     2881 cccaagcaga gagcaactcc atttcatata tatacctacg cacgctcgtt tcacttctag
     2941 cgatggataa cgttagaagc cacacgcagc cgtagatcgc aaaaaagcac cgactcggtg
     3001 ccactttttc aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa
     3061 acaggtcttc tcgaagaccc caagcagaga gcaactccat ttcatatata tacctacgca
     3121 cgctcgtttc acttctagcg atggataacg ttagaagcca cacgcagccg tctcgagaga
     3181 tctaattcag atcactagtg atctataact tcgtataatg tatgctatac gaagttatga
     3241 cgtcattcaa ttagagacta attcaattag agctaattca attagcgccg gagtataaat
     3301 agaggcgctt cgtctacgga gcgacaattc aattcaaaca agcaaagtga acacgtcgct
     3361 aagcgaaagc taagcaaata aacaagcgca gctgaacaag ctaaacaatc ggcccatggt
     3421 gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca
     3481 catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
     3541 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc
     3601 ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc
     3661 cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat
     3721 gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga
     3781 gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca
     3841 gaagaaaacc atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct
     3901 gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt
     3961 caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat
     4021 caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc
     4081 cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtaagcgc cgcgactctg
     4141 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
     4201 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
     4261 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4321 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttagct ggatcttata
     4381 aaaaattcaa ttatacttgc taaactaacg gttaatcgtc gagtggtacc ataacttcgt
     4441 ataatgtatg ctatacgaag ttatgcatgc ggtggatcta aacgagtttt taagcaaact
     4501 cactccctga caataaaaac gcaattatct ttaaataaat atattctatt tattaaccaa
     4561 gtagctaata tagctaaaat caatttgttg ctaacttaaa actaacataa ttatgtatta
     4621 gttatttaca ttcttttggt tggttacaac caaataaata atatcacttt taaaaactta
     4681 agccaggaac tgaataattt tctatttggc tttagtcgac ggtatcgata gacgtcatca
     4741 ctgttcgtat gtattaatgt cggagaagtt acatccagga acgaatccca gattggtcat
     4801 aaactgtggg tcgacatcca cagcgtccgt gtattcaatg ctagccaaat cgtcaaccat
     4861 ggcatcgtaa tcaacggtcg agtaaccaaa gttcgccatt ggaaccagat tcgagtggta
     4921 cggcgggtga ttggtcgtgc tgtcaacata cggaggaggc ggtcgggggg gggcgccacc
     4981 gggagcgaga gcgacggaag cttcattaag gtgcgtagcc ggtggcgggt aagtagagag
     5041 gataccatgt gtatttacgc ttccataatc gttggtctga gtgatagcat cgagagctgc
     5101 atcggagtta tacggagtgg ggttgggaat accgttgaag cggtcgttga cggggttaat
     5161 gctggtactg agaggctgct ccatcacgtg cggcattatc gaggccgtag ccgtggcggt
     5221 aggagggagc tccaactgac ggaggaggcg ggtaatgctc ttattgacac ggctagtagc
     5281 ccaacgggca tggagtcgct cggcagccgg cgaatcttta ccaacgagca gagctgatcc
     5341 ttgtccggcg tcacggacga ggaggttatg caaggcatct tcggaacgcg cgacattttc
     5401 aaacatccag gcgagggcga gctccagcgt acgaatataa ccggtttgaa ctcccctctt
     5461 tttcggcgaa gcctgataag tgcaggacct tccctgggaa acgcacggga aacacgcagg
     5521 ctgtattccg tcgcatttct ctcgggctgc acggcactgg tcgcatgcgc gggacactcg
     5581 ttgacgcttt gagcgtgctg gttccacgag cagggtcgta gttgtgggtc ccagacccgg
     5641 agtatcgatg ttagtgagat cagcgaacct cgcattattc gaggacgcgg ccgtattggc
     5701 cacgtgtccg tcggcgtttc cgtcggcgtg tccgtcggca tgagcgttag cctcagccgc
     5761 agcgttaagc gttttgcgct tgggtggcat cctagggggc ccggggttct cctccacatc
     5821 gccgcaggtc agcaggctgc cgcggccctc acgcgttacg taccactcga cgattaaccg
     5881 ttagtttagc aagtataatt gaatttttta taagatccag ctaagataca ttgatgagtt
     5941 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     6001 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     6061 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct
     6121 ctacaaatgt ggtatggctg attatgatca gagtcgtact agattatttg tatagttcat
     6181 ccatgccatg tgtaatccca gcagctgtta caaactcaag aaggaccatg tggtctctct
     6241 tttcgttggg atctttcgaa agggcagatt gtgtggacag gtaatggttg tctggtaaaa
     6301 ggacagggcc atcgccaatt ggagtatttt gttgataatg gtctgctagt tgaacgcttc
     6361 catcttcaat gttgtgtcta attttgaagt taactttgat tccattcttt tgtttgtctg
     6421 ccatgatgta tacattgtgt gagttatagt tgtattccaa tttgtgtcca agaatgtttc
     6481 catcttcttt aaaatcaata ccttttaact cgattctatt aacaagggta tcaccttcaa
     6541 acttgacttc agcacgtgtc ttgtagttcc cgtcatcttt gaaaaatata gttctttcct
     6601 ggacataacc ttcgggcatg gcactcttga aaaagtcatg ctgtttcata tgatctgggt
     6661 atcttgaaaa gcattgaaca ccataagtta aagtagtgac aagtgttggc catggaacag
     6721 gtagttttcc agtagtgcaa ataaatttaa gggtaagttt tccgtatgtt gcatcacctt
     6781 caccctctcc actgacagaa aatttgtgcc cattaacatc accatctaat tcaacaagaa
     6841 ttgggacaac tccagtgaaa agttcttctc ctttactcat ggtggcgacc ggtggatcgt
     6901 accgtcgact ctagcggtac cccgattgtt tagcttgttc agctgcgctt gtttatttgc
     6961 ttagctttcg cttagcgacg tgttcacttt gcttgtttga attgaattgt cgctccgtag
     7021 acgaagcgcc tctatttata ctccggcggt cgagggttcg aaatcgataa gcttggatcc
     7081 taattgaatt agctctaatt gaattagtct ctaattgaat tagatccccg ggcgagctcg
     7141 aattaaccat tgtgggaaca ctagaactag tggatccccc gggctgcagg aattcgataa
     7201 aagttttgtt actttataga agaaattttg agtttttgtt ttttttaata aataaataaa
     7261 cataaataaa ttgtttgttg aatttattat tagtatgtaa gtgtaaatat aataaaactt
     7321 aatatctatt caaattaata aataaacctc gatatacaga ccgataaaac acatgcgtca
     7381 attttacaca tgattatctt taacgtacgt cacaatatga ttatctttct agggttaatc
     7441 tagctgcgtg ttctgcagcg tgtcgagcat cttcatctgc tccatcacgc tgtaaaacac
     7501 atttgcaccg cgagtctgcc cgtcctccac gggttcaaaa acgtgaatga acgaggcgcg
     7561 ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     7621 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     7681 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
     7741 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     7801 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     7861 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     7921 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     7981 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     8041 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     8101 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     8161 attaacgctt acaatttag
//
