LOCUS       sequence-218856-        8467 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 8467)
  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 8467)
  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+Sxl].1046B
FEATURES             Location/Qualifiers
     source          1..8467
                     /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        5420..5840
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        5861..5936
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    5991..6060
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        complement(6279..6297)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6318..6334)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6318..6334)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6331..6353)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6342..6358
                     /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(6366..6372,6373..6390,6391..6396))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6411..6432
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6549..6566)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6720..7308)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6800..6819)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7479..8270,8271..8339))
                     /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     8102..8121
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8340..8444)
                     /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 acaatttcca ttcgccattc aggctgcgca
      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 cggattgtca actacttgcc
     5281 ccgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     5341 gtggcaccga gtcggtgctt ttttttttgg tacgggaatt gggaattggg caatatttaa
     5401 atggcggcgc gccgaattct tttttgctca cctgtgattg ctcctactca aatacaaaaa
     5461 catcaaattt tctgtcaata aagcatattt atttatattt attttacagg aaagaattcc
     5521 ttttaaagtg tattttaacc tataatgaaa aacgattaaa aaaaatacat aaaataattc
     5581 gaaaattttt gaatagccca ggttgataaa aattcatttc atacgtttta taacttatgc
     5641 ccctaagtat tttttgacca tagtgtttca attctacatt aattttacag agtagaatga
     5701 aacgccacct actcagccaa gaggcgaaaa ggttagctcg ccaagcagag agggcgccag
     5761 tgctcactac tttttataat tctcaacttc tttttccaga ctcagttcgt atatatagac
     5821 ctattttcaa tttaacgtcg cctgagtgtg catcagctgg gttttagagc tagaaatagc
     5881 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     5941 ttttttggta cctctagaga tccactagtg tcgacgatgt aggtcacggt ctcgaagccg
     6001 cggtgcgggt gccagggcgt gcccttgggc tccccgggcg cgtactccac ctcacccatc
     6061 tggtccatca tgatgaacgg gtcgaggtgg cggtagttga tcccggcgaa cgcgcggcgc
     6121 accgggaagc cctcgccctc gaaaccgctg ggcgcggtgg tcacggtgag cacgggacgt
     6181 gcgacggcgt cggcgggtgc ggatacgcgg ggcagcgtca gcgggttctc gacggtcacg
     6241 gcgggcatgt cgacactagt tctagccagc ttttgttccc tttagtgagg gttaatttcg
     6301 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     6361 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     6421 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     6481 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     6541 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     6601 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     6661 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     6721 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     6781 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     6841 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     6901 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     6961 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     7021 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     7081 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     7141 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     7201 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     7261 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     7321 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     7381 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     7441 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     7501 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     7561 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     7621 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     7681 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     7741 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     7801 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     7861 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     7921 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     7981 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     8041 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     8101 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     8161 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     8221 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     8281 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     8341 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     8401 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     8461 gtgccac
//
