LOCUS       sequence-249731-        7994 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses gRNA in Aedes aegypti.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7994)
  AUTHORS   Li M, Yang T, Kandul NP, Bui M, Gamez S, Raban R, Bennett J, Sanchez
            C HM, Lanzaro GC, Schmidt H, Lee Y, Marshall JM, Akbari OS
  TITLE     Development of a confinable gene drive system in the human disease 
            vector Aedes aegypti.
  JOURNAL   Elife. 2020 Jan 21;9. pii: 51701. doi: 10.7554/eLife.51701.
  PUBMED    31960794
REFERENCE   2  (bases 1 to 7994)
  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: "2020-01-21"; volume: "9. pii: 51701. doi"; pages: " 
            10.7554/eLife.51701"
COMMENT     Sequence Label: U6a-T
FEATURES             Location/Qualifiers
     source          1..7994
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1..3,4..6,7..720)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(46..67)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(307..326)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     654..675
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    843..964
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(880..899)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     934..953
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    2104..2173
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     repeat_region   3026..3060
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3241..3257)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3241..3257)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3254..3276)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3265..3281
                     /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(3289..3295,3296..3313,3314..3319))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3334..3355
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3472..3489)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3643..4231)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3723..3742)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4402..5193,5194..5262))
                     /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     5025..5044
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5263..5367)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5435..5453
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5491..5513)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5613..5632
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5826..5848
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5840..5857
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5841..5857
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   6561..6623
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     regulatory      join(7989..7994,1..4)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
       61 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      121 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      181 ctcgtgacca ccctgacctg gggcgtgcag tgcttcagcc gctaccccga ccacatgaag
      241 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
      301 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      361 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      421 aagctggagt acaactacat cagccacaac gtctatatca ccgccgacaa gcagaagaac
      481 ggcatcaagg ccaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
      541 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
      601 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
      661 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
      721 agcggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
      781 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
      841 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
      901 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
      961 cttaaagctt gggccccact ttccaaaatt catttggtaa tttctacaag aattttttca
     1021 agaaatttct taagaatttt tgaaaaaaaa aatggtaatt tttactgaat tgataaacgc
     1081 aattgatagg taaaccattg ctaaatatag tcattcacca aacacactgc ttttttctcg
     1141 tctgaaaatt ttttagttgt ttcgcaagta acccatttta tatccaaaaa ttactttctg
     1201 tacatttttc tcgctctttc ttagcgaatg gattattgat ttctaagctt gtcattgact
     1261 tgactacttt tattgaactg ataattctat taacagaaat taataacaac agtttgctta
     1321 tgaaataaca tgatagccaa gtcaataaca ggtccagttt agactttcgg gttttccata
     1381 gtaaatatac tacaaataat attaatgttc catgaaaaaa ggagtaagag tctggtaacc
     1441 ctagtgcacg caaatatctc gcgggcatat ttggttgctg aggtatattt atatttgaac
     1501 gccatgagaa aaagcggaag aaattggctc atggccgatt ttaaggatat ttaaaaattg
     1561 tacaatgtac atataatagg ccaggagaag tggatgaact tgtcattcat ttttctgtca
     1621 attctcatac aaaatctact ttttctttga cataaattca ctctaggtga accacttccc
     1681 ctggcctatt aaacatccgt tccttcaatg tgttcttttt tttaagcgtg tgttaaaagt
     1741 ttgctctgct ggtgaattca cgctctaccc gttcaggcag cattcatcga aaagccctat
     1801 ctgctcgcac acatttacaa aatgctgatt gcgttgtgtg ctgaatgggt cactcgtccg
     1861 tcactgcttg ctgtgtacac tgtacagtta cgcagtctgt gcatcgctag aatcatattt
     1921 acggaagagt attatatata cccgatgcgt tgctcttcga ttggtgcgat cttgaccccc
     1981 gggtcttaga gctatgctgg aaacagcata gcaagttaag ataaggctag tccgttatca
     2041 acttgaaaaa gtggcaccga gtcggtgctt ttttggcgcg ccgtcgacga tgtaggtcac
     2101 ggtctcgaag ccgcggtgcg ggtgccaggg cgtgcccttg ggctccccgg gcgcgtactc
     2161 cacctcaccc atctggtcca tcatgatgaa cgggtcgagg tggcggtagt tgatcccggc
     2221 gaacgcgcgg cgcaccggga agccctcgcc ctcgaaaccg ctgggcgcgg tggtcacggt
     2281 gagcacggga cgtgcgacgg cgtcggcggg tgcggatacg cggggcagcg tcagcgggtt
     2341 ctcgacggtc acggcgggca tgtcgacggc cggccgatct cggatctgac aatgttcagt
     2401 gcagagactc ggctacgcct cgtggacttt gaagttgacc aacaatgttt attcttacct
     2461 ctaatagtcc tctgtggcaa ggtcaagatt ctgttagaag ccaatgaaga acctggttgt
     2521 tcaataacat tttgttcgtc taatatttca ctaccgcttg acgttggctg cacttcatgt
     2581 acctcatcta taaacgcttc ttctgtatcg ctctggacgt catcttcact tacgtgatct
     2641 gatatttcac tgtcagaatc ctcaccaaca agctcgtcat cgctttgcag aagagcagag
     2701 aggatatgct catcgtctaa agaactaccc attttattat atattagtca cgatatctat
     2761 aacaagaaaa tatatatata ataagttatc acgtaagtag aacatgaaat aacaatataa
     2821 ttatcgtatg agttaaatct taaaagtcac gtaaaagata atcatgcgtc attttgactc
     2881 acgcggtcgt tatagttcaa aatcagtgac acttaccgca ttgacaagca cgcctcacgg
     2941 gagctccaag cggcgactga gatgtcctaa atgcacagcg acggattcgc gctatttaga
     3001 aagagagagc aatatttcaa gaatgcatgc gtcaatttta cgcagactat ctttctaggg
     3061 ttaaaaaaga tttgcgcttt actcgaccta aactttaaac acgtcataga atcttcgttt
     3121 gacaaaaacc acattgtggc caagctgtgt gacgcgacgc gcgctaaaga atggcaaacc
     3181 aagtcgcgcg agcgtcgact ctagaggatc cccgggtacc gagctcgaat tcgtaatcat
     3241 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     3301 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     3361 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     3421 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     3481 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     3541 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     3601 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     3661 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     3721 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     3781 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     3841 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3901 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3961 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4021 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4081 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4141 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4201 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4261 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4321 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     4381 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     4441 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     4501 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     4561 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     4621 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     4681 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     4741 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     4801 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     4861 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     4921 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     4981 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     5041 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     5101 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     5161 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5221 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     5281 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     5341 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     5401 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     5461 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     5521 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     5581 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     5641 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     5701 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     5761 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     5821 gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagctttgtt taaaaatata
     5881 acaaaattgt gatcccacaa aatgaagtgg ggcaaaatca aataattaat agtgtccgta
     5941 aacttgttgg tcttcaactt tttgaggaac acgttggacg gcaaatccgt gactataaca
     6001 caagttgatt taataatttt agccaacacg tcgggctgcg tgttttttgc cgacgcgtct
     6061 gtgtacacgt tgattaactg gtcgattaaa ctgttgaaat aatttaattt ttggttcttc
     6121 tttaaatctg tgatgaaatt ttttaaaata actttaaatt cttcattggt aaaaaatgcc
     6181 acgttttgca acttgtgagg gtctaatatg aggtcaaact cagtaggagt tttatccaaa
     6241 aaagaaaaca tgattacgtc tgtacacgaa cgcgtattaa cgcagagtgc aaagtataag
     6301 agggttaaaa aatatatttt acgcaccata tacgcatcgg gttgatatcg ttaatatgga
     6361 tcaatttgaa cagttgatta acgtgtctct gctcaagtct ttgatcaaaa cgcaaatcga
     6421 cgaaaatgtg tcggacaata tcaagtcgat gagcgaaaaa ctaaaaaggc tagaatacga
     6481 caatctcaca gacagcgttg agatatacgg tattcacgac agcaggctga ataataaaaa
     6541 aattagaaac tattatttaa ccctagaaag ataatcatat tgtgacgtac gttaaagata
     6601 atcatgcgta aaattgacgc atgtgtttta tcggtctgta tatcgaggtt tatttattaa
     6661 tttgaataga tattaagttt tattatattt acacttacat actaataata aattcaacaa
     6721 acaatttatt tatgtttatt tatttattaa aaaaaaacaa aaactcaaaa tttcttctat
     6781 aaagtaacaa aacttttaaa cattctctct tttacaaaaa taaacttatt ttgtacttta
     6841 aaaacagtca tgttgtatta taaaataagt aattagctta acttatacat aatagaaaca
     6901 aattatactt attagtcagt cagaaacaac tttggcacat atcaatatta tgctctcgac
     6961 aaataacttt tttgcatttt ttgcacgatg catttgcctt tcgccttatt ttagaggggc
     7021 agtaagtaca gtaagtacgt tttttcatta ctggctcttc agtactgtca tctgatgtac
     7081 caggcacttc atttggcaaa atattagaga tattatcgcg caaatatctc ttcaaagtag
     7141 gagcttctaa acgcttacgc ataaacgatg acgtcaggct catgtaaagg tttctcataa
     7201 attttttgcg actttgaacc ttttctccct tgctactgac attatggctg tatataataa
     7261 aagaatttat gcaggcaatg tttatcattc cgtacaataa tgccataggc cacctattcg
     7321 tcttcctact gcaggtcatc acagaacaca tttggtctag cgtgtccact ccgcctttag
     7381 tttgattata atacataacc atttgcggtt taccggtact ttcgttgata gaagcatcct
     7441 catcacaaga tgataataag tataccatct tagctggctt cggtttatat gagacgagag
     7501 taaggggtcc gtcaaaacaa aacatcgatg ttcccactgg cctggagcga ctgtttttca
     7561 gtacttccgg tatctcgcgt ttgtttgatc gcacggttcc cacaatggtt aattcgagct
     7621 cgcccgggct taagagttcc cacaatggtt aattcgagct cgcccgggct taagagttcc
     7681 cacaatggtt aattcgagct cgcccgggct taagagttcc cacaatggtt aattcgagct
     7741 cgcccgggga tctaattcaa ttagagacta attcaattag agctaattca attaggatcc
     7801 aagcttatcg atttcgaacc ctcgaccgcc ggagtataaa tagaggcgct tcgtctacgg
     7861 agcgacaatt caattcaaac aagcaaagtg aacacgtcgc taagcgaaag ctaagcaaat
     7921 aaacaagcgc agctgaacaa gctaaacaat cggggtaccg ctagagtcga cggtacgatc
     7981 caccggtcgc cacc
//
