LOCUS       sequence-361369-        6785 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  donor plasmid for CRISPR Cas9 knockin near the Aedes aegypti m / M 
            locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6785)
  AUTHORS   Lutrat C, Burckbuchler M, Olmo RP, Beugnon R, Fontaine A, Akbari OS,
            Argiles-Herrero R, Baldet T, Bouyer J, Marois E
  TITLE     Combining two genetic sexing strains allows sorting of 
            non-transgenic males for Aedes genetic control.
  JOURNAL   Commun Biol. 2023 Jun 16;6(1):646. doi: 10.1038/s42003-023-05030-7.
  PUBMED    37328568
REFERENCE   2  (bases 1 to 6785)
  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: "Commun 
            Biol."; date: "2023-06-16"; volume: "6(1):646. doi"; pages: " 
            10.1038/s42003-023-05030-7"
COMMENT     Sequence Label: pX4
FEATURES             Location/Qualifiers
     source          1..6785
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(603..727)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(join(1922..2635,2636..2638,2639..2641))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1925..2635,2636..2638,2639..2641))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1967..1988)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2316..2335
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2575..2596
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5026..5045)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5027..5045)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5050..5066)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5050..5066)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5168..5187)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5179..5988
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5246..5265)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6135..6723
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6624..6643
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccct acaggtcact aataccatct aagtagttga
      601 ttcatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt ttatgcaaaa
      661 tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcaact tttctataca
      721 aagttggtac cgggcccccc gctagcgtcg acggtatcga taagcttgat cggatccgct
      781 ccgttgattg atgaatcatt gattcagtaa cttagttgat caataactct aacacgaaaa
      841 ttacggatat aatttcgtaa tttcgaattt tgagggtttt gttagatgaa ctttgattta
      901 agatcttgtt tcgtaggaag ataaatgtca caatcattgt tttgcaatac cctattattg
      961 atataaaaaa tctgaaattt ttttaaattt agtttatagg cactcgttgt ttacgaaata
     1021 tgatgcaaaa ttcagtttat tataaaaaag ttttttattt ttcgaattag ggtagcatcc
     1081 caaattatgt cacgctaaat ttcaactttt tcgaccccct tccctcctct actttgtcac
     1141 attttttgta tgagtcctcc gaaaattttg taaggcttga ccccctccac ccctcttaaa
     1201 gcgtgacgta atttgtgcat gaccccttag atagaaatgt tgaaaaccta tcaacataaa
     1261 tctgggcaaa acttcaatca aaataaatgc aatgtacact gaacaggtaa gtgtaggagg
     1321 taagaaaaga tagctaaaaa aaactcgact cgacttgatg atggttttga aaaaactcaa
     1381 aaacgacatg taacaataaa ggtaagtatt gaatggatac ttgtatacaa agtactttga
     1441 agttcaattc ttgttgaaat tgaactgtct tccataacat ttattcttaa acactaaatt
     1501 tctaagtttt aacggcattt cgtggtgcgc actgaacttg ttctcttttc tcctgataca
     1561 taactcacat gtagctctga tgttgaaaca ctttcatatc tatcattaag tcagcaatct
     1621 caggacaatt ttcatcatat tttttctctt gtattttttc aacaggttat ctacattcgt
     1681 cccctgatga gtacgttcaa tattatggac cacaaactca cacatacacg cttcccaggt
     1741 aagttcctat acgaaacccc aacaaaaacc ataattgttt agacttgtga acaaaattgg
     1801 atccgacttt attgattacg ttgttaagag aacaaatctt ttacaactga attcatttgt
     1861 tctcgtttca ttttttttcg caaaacattg atcgagaatt cgattgattt ccgattcgaa
     1921 tttacttgta cagctcgtcc atgccgagag tgatcccggc ggcggtcacg aactccagca
     1981 ggaccatgtg atcgcgcttc tcgttggggt ctttgctcag ggcggactgg gtgctcaggt
     2041 agtggttgtc gggcagcagc acggggccgt cgccgatggg ggtgttctgc tggtagtggt
     2101 cggcgagctg cacgctgccg tcctcgatgt tgtggcggat cttgaagttc accttgatgc
     2161 cgttcttctg cttgtcggcc atgatataga cgttgtggct gttgtagttg tactccagct
     2221 tgtgccccag gatgttgccg tcctccttga agtcgatgcc cttcagctcg atgcggttca
     2281 ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga
     2341 agaagatggt gcgctcctgg acgtagcctt cgggcatggc ggacttgaag aagtcgtgct
     2401 gcttcatgtg gtcggggtag cggctgaagc actgcacgcc gtaggtcagg gtggtcacga
     2461 gggtgggcca gggcacgggc agcttgccgg tggtgcagat gaacttcagg gtcagcttgc
     2521 cgtaggtggc atcgccctcg ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc
     2581 cgtccagctc gaccaggatg ggcaccaccc cggtgaacag ctcctcgccc ttgctcacca
     2641 tggttgaaat ctctgtttag cagaaaaaga aacgaggaaa cgcttcagta attggttgtg
     2701 aaatgcaaac tctcatttga tattgattca ttgcctttgg cttcgagcac gacacgacag
     2761 gttttaaact tgttttgctt tgtctgcgtt tgcagtcgca ggccaagtga aaaatataca
     2821 cttgaaggtg atgacgtcac aacaacgccc tactttttag tgaaaaatta acttgttttc
     2881 gactttgaac tacgtagttt ggaaattgcg tatcttcaag ttttcacgat ttcctcaagg
     2941 tttttctcga tatgtgttaa tattacctta atgggtaatt atcaccacaa tatttaattt
     3001 tagaaatatg gcgaattcgc gcttgctcta gaaatttaat tcaattagca gcgatgattc
     3061 aacgaaatat gattatcgcc gtgaatcaca attgggtttt atcaatgatg atgaaactgc
     3121 gttgcaaatt ttcactaatc actcaaagct caatagtcgc catcttggaa aatagtttgc
     3181 tcattcaaag acaaaggaat cattcaccaa actagttttc gctcatagct ataatttcat
     3241 catttaattt acctaccttc actagaagat tcccttcacc gaaatgcact ttcacaaatt
     3301 taatagtaat tgtcctttga acaaagcttt gttcactctg aaattttctc ctctggctaa
     3361 ttggatcact ctttttcact agagacttca cttcacttgc actggcactg cttacttgga
     3421 ccgcgtaatg ttcactccac taggaaacgt attcgattga gctggttttg cctttgcagg
     3481 ggcgttttat agacactgcc gtagtggtgg ttgtacttct agaaaatttc tgcaatccat
     3541 tcatacatat acctctgttc ggttggatgg ctctaaatcg atcgactatg ggtaccatcc
     3601 ctgtgtctga atggacagca aaacgtgctt gtgtctgtta gtcgttcatt ctgtaccttg
     3661 aacgatgcag ttcaacttct ggcaaagacg tcaatgtacc taccattcgt gtatatggca
     3721 tacagagaaa gatgtgcgaa tgcctttttc gatagagaaa ggatttcctg atttgatcga
     3781 caatttcggt agctattctt agcttcgaat gtaatctgat aaccaaatcc agagacaaag
     3841 cagtaatttg ggaatttcac ttgaatcatt ctaattgtat catttgctgt attagtgcag
     3901 tcagcaaagt gacgtcaacc cttctaaatc gatatacttc tgggaagctt tctttcttgt
     3961 ctggctcagc tggtgccaag gcaaattata attggattca atgcacaagc tacatgtaaa
     4021 gatcttttct acgcctaccc cctcggacaa caggcgtccc atgattcatc aatcaacgga
     4081 gcgacctcat agaaatatta cgccaactgg aataccaatg gcatccagac atatctatgg
     4141 gtatgacgcc caagttgaca ataatgcaag cacattccac cggaacaacg atatgtactg
     4201 gaccttgcaa actcgtcgtc gaccaattcc aatggaccag ccgaatatgc gtgaagctct
     4261 taatttatcc cagcagagcg ttagaccaat agaagaactc tattcaacac caaacaagat
     4321 caaatcaaat gcactgaaaa ctacaccaac tgggtttgct acgtactttt ctcctatcgc
     4381 taaaagtaac acgggtggtg aattaaacga cgatgtggat agcagtttta agatttctcc
     4441 gatcgaccct cggagtattg ctcatttcaa aacatctact ccgtcaaaac caaacgagac
     4501 atcactttct cctacaagat cgcttaccga agagcttcga tatagattaa gactgcaaca
     4561 ggtcggagtt cgatcacatg ggaattctcc ggtaagctcc ggccgttcaa caccaaaaaa
     4621 cgtacttgaa gcacaagctc cacgtggcag acacagttgg gcttccaaca gtaccgacat
     4681 tccacagaca tgctctgatc gacttgggac accaaaaacg agccttatgg atttcaaaaa
     4741 gttgcttctt gcgcatggat cgaaatctaa tatttcgtct ggaagtaaaa tttctgccgt
     4801 tgaactactt aagaaatcaa aaagtaacgc tccagttaac ccagtttctc cagttacaaa
     4861 aacttctgca aatagtagtt tgaatatttt ggacctttcg ggatctccaa aaacattcgc
     4921 tacaagacgc atgattcgac aaggaaactt tggaaatggt tcaccatcaa agctcggtaa
     4981 tgtgtctaaa cattcttcaa ggggtggttg gcggtacagc ttatccccta tagtgagtcg
     5041 tattacatgg tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt
     5101 tacattgcac aagataaaaa tatatcatca tgaacaataa aactgtctgc ttacataaac
     5161 agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcgaggcc gcgattaaat
     5221 tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca
     5281 ggtgcgacaa tctatcgctt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat
     5341 ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg
     5401 gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta
     5461 ctcaccactg cgatccccgg aaaaacagca ttccaggtat tagaagaata tcctgattca
     5521 ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt
     5581 tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg
     5641 aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa
     5701 caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat
     5761 ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat
     5821 gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc
     5881 ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct
     5941 gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt
     6001 aattggttgt aacactggca gagcattacg ctgacttgac gggacggcgc aagctcatga
     6061 ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc cgtagaaaag
     6121 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     6181 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     6241 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     6301 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     6361 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     6421 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     6481 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     6541 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     6601 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     6661 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     6721 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     6781 atgtt
//
