LOCUS       sequence-218860-        8468 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express two gRNA targeting bTub.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8468)
  AUTHORS   Kandul NP, Liu J, Sanchez C HM, Wu SL, Marshall JM, Akbari OS
  TITLE     Transforming insect population control with precision guided sterile
            males with demonstration in flies.
  JOURNAL   Nat Commun. 2019 Jan 8;10(1):84. doi: 10.1038/s41467-018-07964-7.
  PUBMED    30622266
REFERENCE   2  (bases 1 to 8468)
  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: "Nat 
            Commun."; date: "2019-01-8"; volume: "10(1):84. doi"; pages: " 
            10.1038/s41467-018-07964-7"
COMMENT     Sequence Label: gRNAs[bTub+Tra].1046D
FEATURES             Location/Qualifiers
     source          1..8468
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      2..457
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(89..108)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     299..320
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     583..605
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     597..614
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     598..614
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     621..640
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        621..639
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(673..691)
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     CDS             join(1143..1214,1616..1889,1964..2618,2680..2995,3199..3330,
                     3401..4015)
                     /codon_start=1
                     /gene="white"
                     /product="Drosophila white gene eye color pigment"
                     /label=mini-white
                     /note="This is a modified version of the white gene lacking
                     part of the first intron."
                     /translation="MGQEDQELLIRGGSKHPSAEHLNNGDSGAASQSCINQGFGQAKNY
                     GTLRPPSPPEDSGSGSGQLAENLTYAWHNMDIFGAVNQPGSGWRQLVNRTRGLFCNERH
                     IPAPRKHLLKNVCGVAYPGELLAVMGSSGAGKTTLLNALAFRSPQGIQVSPSGMRLLNG
                     QPVDAKEMQARCAYVQQDDLFIGSLTAREHLIFQAMVRMPRHLTYRQRVARVDQVIQEL
                     SLSKCQHTIIGVPGRVKGLSGGERKRLAFASEALTDPPLLICDEPTSGLDSFTAHSVVQ
                     VLKKLSQKGKTVILTIHQPSSELFELFDKILLMAEGRVAFLGTPSEAVDFFSYVGAQCP
                     TNYNPADFYVQVLAVVPGREIESRDRIAKICDNFAISKVARDMEQLLATKNLEKPLEQP
                     ENGYTYKATWFMQFRAVLWRSWLSVLKEPLLVKVRLIQTTMVAILIGLIFLGQQLTQVG
                     VMNINGAIFLFLTNMTFQNVFATINVFTSELPVFMREARSRLYRCDTYFLGKTIAELPL
                     FLTVPLVFTAIAYPMIGLRAGVLHFFNCLALVTLVANVSTSFGYLISCASSSTSMALSV
                     GPPVIIPFLLFGGFFLNSGSVPVYLKWLSYLSWFRYANEGLLINQWADVEPGEISCTSS
                     NTTCPSSGKVILETLNFSAADLPLDYVGLAILIVSFRVLAYLALRLRARRKE"
     protein_bind    4790..4823
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4842..5262
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        5283..5358
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        5421..5841
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        5862..5937
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    5992..6061
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        complement(6280..6298)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6319..6335)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6319..6335)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6332..6354)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6343..6359
                     /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(6367..6373,6374..6391,6392..6397))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6412..6433
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6550..6567)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6721..7309)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6801..6820)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7480..8271,8272..8340))
                     /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     8103..8122
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8341..8445)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
       61 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
      121 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
      181 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
      241 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
      301 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
      361 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
      421 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggstgcgca
      481 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
      541 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
      601 aaacgacggc cagtgaattg taatacgact cactataggg cgaattgggt acgtaccggg
      661 cccctagtat gtatgtaagt taataaaacc catttttgcg gaaagtagat aaaaaaaaca
      721 tttttttttt ttactgcact ggatatcatt gaacttatct gatcagtttt aaatttactt
      781 cgatccaagg gtatttgatg taccaggttc tttcgattac ctctcactca aaatgacatt
      841 ccactcaaag tcagcgctgt ttgcctcctt ctctgtccac agaaatatcg ccgtctcttt
      901 cgccgctgcg tccgctatct ctttcgccac cgtttgtagc gttacgtagc gtcaatgtcc
      961 gccttcagtt gcattttgtc agcggtttcg tgacgaagct ccaagcggtt tacgccatca
     1021 attaaacaca aagtgctgtg ccaaaactcc tctcgcttct tatttttgtt tgttttttga
     1081 gtgattgggg tggtgattgg ttttgggtgg gtaagcaggg gaaagtgtga aaaatcccgg
     1141 caatgggcca agaggatcag gagctattaa ttcgcggagg cagcaaacac ccatctgccg
     1201 agcatctgaa caatgtgagt agtacatgtg catacatctt aagttcactt gatctatagg
     1261 aactgcgatt gcaacatcaa attgtctgcg gcgtgagaac tgcgacccac aaaaatccca
     1321 aaccgcaatt gcacaaacaa atagtgacac gaaacagatt attctggtag ctgttctcgc
     1381 tatataagac aatttttgag atcatatcat gatcaagaca tctaaaggca ttcattttcg
     1441 actatattct tttttacaaa aaatataaca accagatatt ttaagctgat cctagatgca
     1501 caaaaaataa ataaaagtat aaacctactt cgtaggatac ttcggggtac tttttgttcg
     1561 gggttagatg agcataacgc ttgtagttga tatttgagat cccctatcat tgcagggtga
     1621 cagcggagcg gcttcgcaga gctgcattaa ccagggcttc gggcaggcca aaaactacgg
     1681 cacgctccgg ccacccagtc cgccggagga ctccggttca gggagcggcc aactagccga
     1741 gaacctcacc tatgcctggc acaatatgga catctttggg gcggtcaatc agccgggctc
     1801 cggatggcgg cagctggtca accggacacg cggactattc tgcaacgagc gacacatacc
     1861 ggcgcccagg aaacatttgc tcaagaacgg tgagtttcta ttcgcagtcg gctgatctgt
     1921 gtgaaatctt aataaagggt ccaattacca atttgaaact cagtttgcgg cgtggcctat
     1981 ccgggcgaac ttttggccgt gatgggcagt tccggtgccg gaaagacgac cctgctgaat
     2041 gcccttgcct ttcgatcgcc gcagggcatc caagtatcgc catccgggat gcgactgctc
     2101 aatggccaac ctgtggacgc caaggagatg caggccaggt gcgcctatgt ccagcaggat
     2161 gacctcttta tcggctccct aacggccagg gaacacctga ttttccaagc catggtgcgg
     2221 atgccacgac atctgaccta tcggcagcga gtggcccgcg tggatcaggt gatccaggag
     2281 ctttcgctca gcaaatgtca gcacacgatc atcggtgtgc ccggcagggt gaaaggtctg
     2341 tccggcggag aaaggaagcg tctggcattc gcctccgagg ctctaaccga tccgccgctt
     2401 ctgatctgcg atgagcccac ctccggactg gactccttta ccgcccacag cgtcgtccag
     2461 gtgctgaaga agctgtcgca gaagggcaag accgtcatcc tgaccattca tcagccgtct
     2521 tccgagctgt ttgagctctt tgacaagatc cttctgatgg ccgagggcag ggtagctttc
     2581 ttgggcactc ccagcgaagc cgtcgacttc ttttcctagt gagttcgatg tgtttattaa
     2641 gggtatctag tattacataa catctcaact cctatccagc gtgggtgccc agtgtcctac
     2701 caactacaat ccggcggact tttacgtaca ggtgttggcc gttgtgcccg gacgggagat
     2761 cgagtcccgt gatcggatcg ccaagatatg cgacaatttt gccattagca aagtagcccg
     2821 ggatatggag cagttgttgg ccaccaaaaa tctggagaag ccactggagc agccggagaa
     2881 tgggtacacc tacaaggcca cctggttcat gcagttccgg gcggtcctgt ggcgatcctg
     2941 gctgtcggtg ctcaaggaac cactcctcgt aaaagtgcga cttattcaga caacggtgag
     3001 tggttccagt ggaaacaaat gatataacgc ttacaattct tggaaacaaa ttcgctagat
     3061 tttagataga attgcctgat tccacaccct tcttagtttt tttcaatgag atgtatagtt
     3121 tatagttttg cagaagataa ataaatttca tttaactcgc gaatattaat gagatgcgag
     3181 taacatttta atttgcagat ggttgccatc ttgattggcc tcatcttttt gggccaacaa
     3241 ctcacgcaag tgggtgtgat gaatatcaac ggagccatct tcctcttcct gaccaacatg
     3301 acctttcaaa acgtctttgc cacgataaat gtaagtcatg tttagaatac atttgcattt
     3361 caataattta ctaactttct aatgaatcga ttcgatttag gtgttcacct cagagctgcc
     3421 agtttttatg agggaggccc gaagtcgact ttatcgctgt gacacatact ttctgggcaa
     3481 aacgattgcc gaattgccgc tttttctcac agtgccactg gtcttcacgg cgattgccta
     3541 tccgatgatc ggactgcggg ccggagtgct gcacttcttc aactgcctgg cgctggtcac
     3601 tctggtggcc aatgtgtcaa cgtccttcgg atatctaata tcctgcgcca gctcctcgac
     3661 ctcgatggcg ctgtctgtgg gtccgccggt tatcatacca ttcctgctct ttggcggctt
     3721 cttcttgaac tcgggctcgg tgccagtata cctcaaatgg ttgtcgtacc tctcatggtt
     3781 ccgttacgcc aacgagggtc tgctgattaa ccaatgggcg gacgtggagc cgggcgaaat
     3841 tagctgcaca tcgtcgaaca ccacgtgccc cagttcgggc aaggtcatcc tggagacgct
     3901 taacttctcc gccgccgatc tgccgctgga ctacgtgggt ctggccattc tcatcgtgag
     3961 cttccgggtg ctcgcatatc tggctctaag acttcgggcc cgacgcaagg agtagccgac
     4021 atatatccga aataactgct tgtttttttt ttttaccatt attaccatcg tgtttactgt
     4081 ttattgcccc ctcaaaaagc taatgtaatt atatttgtgc caataaaaac aagatatgac
     4141 ctatagaata caagtatttc cccttcgaac atccccacaa gtagactttg gatttgtctt
     4201 ctaaccaaaa gacttacaca cctgcatacc ttacatcaaa aactcgttta tcgctacata
     4261 aaacaccggg atatattttt tatatacata cttttcaaat cgcgcgccct cttcataatt
     4321 cacctccacc acaccacgtt tcgtagttgc tctttcgctg tctcccaccc gctctccgca
     4381 acacattcac cttttgttcg acgaccttgg agcgactgtc gttagttccg cgcgattcgg
     4441 ttcgctcaaa tggttccgag tggttcattt cgtctcaata gaaattagta ataaatattt
     4501 gtatgtacaa tttatttgct ccaatatatt tgtatatatt tccctcacag ctatatttat
     4561 tctaatttaa tattatgact ttttaaggta attttttgtg acctgttcgg agtgattagc
     4621 gttacaattt gaactgaaag tgacatccag tgtttgttcc ttgtgtagat gcatctcaaa
     4681 aaaatggtgg gcataatagt gttgtttata tatatcaaaa ataacaacta taataataag
     4741 aatacattta atttagaaaa tgcttggatt tcactggaac taggctagca taacttcgta
     4801 taatgtatgc tatacgaagt tatgctagcg gatccgaatt cttttttgct cacctgtgat
     4861 tgctcctact caaatacaaa aacatcaaat tttctgtcaa taaagcatat ttatttatat
     4921 ttattttaca ggaaagaatt ccttttaaag tgtattttaa cctataatga aaaacgatta
     4981 aaaaaaatac ataaaataat tcgaaaattt ttgaatagcc caggttgata aaaattcatt
     5041 tcatacgttt tataacttat gcccctaagt attttttgac catagtgttt caattctaca
     5101 ttaattttac agagtagaat gaaacgccac ctactcagcc aagaggcgaa aaggttagct
     5161 cgccaagcag agagggcgcc agtgctcact actttttata attctcaact tctttttcca
     5221 gactcagttc gtatatatag acctattttc aatttaacgt cgcggcgaga aagagaatac
     5281 cagttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     5341 gtggcaccga gtcggtgctt ttttttttgg taccgggaat tgggaattgg gcaatattta
     5401 aatggcggcg cgccgaattc ttttttgctc acctgtgatt gctcctactc aaatacaaaa
     5461 acatcaaatt ttctgtcaat aaagcatatt tatttatatt tattttacag gaaagaattc
     5521 cttttaaagt gtattttaac ctataatgaa aaacgattaa aaaaaataca taaaataatt
     5581 cgaaaatttt tgaatagccc aggttgataa aaattcattt catacgtttt ataacttatg
     5641 cccctaagta ttttttgacc atagtgtttc aattctacat taattttaca gagtagaatg
     5701 aaacgccacc tactcagcca agaggcgaaa aggttagctc gccaagcaga gagggcgcca
     5761 gtgctcacta ctttttataa ttctcaactt ctttttccag actcagttcg tatatataga
     5821 cctattttca atttaacgtc gcctgagtgt gcatcagctg ggttttagag ctagaaatag
     5881 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     5941 tttttttggt acctctagag atccactagt gtcgacgatg taggtcacgg tctcgaagcc
     6001 gcggtgcggg tgccagggcg tgcccttggg ctccccgggc gcgtactcca cctcacccat
     6061 ctggtccatc atgatgaacg ggtcgaggtg gcggtagttg atcccggcga acgcgcggcg
     6121 caccgggaag ccctcgccct cgaaaccgct gggcgcggtg gtcacggtga gcacgggacg
     6181 tgcgacggcg tcggcgggtg cggatacgcg gggcagcgtc agcgggttct cgacggtcac
     6241 ggcgggcatg tcgacactag ttctagccag cttttgttcc ctttagtgag ggttaatttc
     6301 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     6361 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     6421 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     6481 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     6541 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     6601 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     6661 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     6721 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     6781 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     6841 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6901 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6961 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     7021 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     7081 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     7141 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     7201 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     7261 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     7321 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     7381 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     7441 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     7501 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     7561 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     7621 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     7681 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     7741 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     7801 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     7861 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     7921 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     7981 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     8041 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     8101 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     8161 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     8221 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     8281 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     8341 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     8401 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     8461 agtgccac
//
