LOCUS       sequence-301652-        8470 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The attB plasmid with the mini-white harboring two U6.3-gRNAs 
            targeting Dmel tra and bTub genes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8470)
  AUTHORS   Kandul NP, Liu J, Akbari OS
  TITLE     Temperature-Inducible Precision-Guided Sterile Insect Technique.
  JOURNAL   CRISPR J. 2021 Dec;4(6):822-835. doi: 10.1089/crispr.2021.0077. Epub
            2021 Nov 3.
  PUBMED    34735288
REFERENCE   2  (bases 1 to 8470)
  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"; doi: 
            "10.1089/crispr.2021.0077"; journalName: "CRISPR J"; date: 
            "2021-12"; volume: "4"; issue: "6"; pages: "822-835"
COMMENT     Sequence Label: 1046E=dgRNAs[traB,bTub]
FEATURES             Location/Qualifiers
     source          1..8470
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    597..630
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        649..1069
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        1090..1165
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1228..1648
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        1669..1744
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    1799..1868
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        complement(2087..2105)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2126..2142)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2126..2142)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2139..2161)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2150..2166
                     /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(2174..2180,2181..2198,2199..2204))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2219..2240
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2357..2374)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2528..3116)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2608..2627)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3287..4078,4079..4147))
                     /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     3910..3929
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4148..4252)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4278..4733)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4415..4436)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4627..4646
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4860..4882
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4874..4891
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4875..4891
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4898..4917
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4898..4916
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4950..4968)
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     CDS             join(5420..5491,5893..6166,6241..6895,6957..7272,7476..7607,
                     7678..8292)
                     /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"
ORIGIN
        1 ttgtcttcta accaaaagac ttacacacct gcatacctta catcaaaaac tcgtttatcg
       61 ctacataaaa caccgggata tattttttat atacatactt ttcaaatcgc gcgccctctt
      121 cataattcac ctccaccaca ccacgtttcg tagttgctct ttcgctgtct cccacccgct
      181 ctccgcaaca cattcacctt ttgttcgacg accttggagc gactgtcgtt agttccgcgc
      241 gattcggttc gctcaaatgg ttccgagtgg ttcatttcgt ctcaatagaa attagtaata
      301 aatatttgta tgtacaattt atttgctcca atatatttgt atatatttcc ctcacagcta
      361 tatttattct aatttaatat tatgactttt taaggtaatt ttttgtgacc tgttcggagt
      421 gattagcgtt acaatttgaa ctgaaagtga catccagtgt ttgttccttg tgtagatgca
      481 tctcaaaaaa atggtgggca taatagtgtt gtttatatat atcaaaaata acaactataa
      541 taataagaat acatttaatt tagaaaatgc ttggatttca ctggaactag gctagcataa
      601 cttcgtataa tgtatgctat acgaagttat gctagcggat ccgaattctt ttttgctcac
      661 ctgtgattgc tcctactcaa atacaaaaac atcaaatttt ctgtcaataa agcatattta
      721 tttatattta ttttacagga aagaattcct tttaaagtgt attttaacct ataatgaaaa
      781 acgattaaaa aaaatacata aaataattcg aaaatttttg aatagcccag gttgataaaa
      841 attcatttca tacgttttat aacttatgcc cctaagtatt ttttgaccat agtgtttcaa
      901 ttctacatta attttacaga gtagaatgaa acgccaccta ctcagccaag aggcgaaaag
      961 gttagctcgc caagcagaga gggcgccagt gctcactact ttttataatt ctcaacttct
     1021 ttttccagac tcagttcgta tatatagacc tattttcaat ttaacgtcgg attccgtact
     1081 ttgcagacgg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
     1141 tgaaaaagtg gcaccgagtc ggtgcttttt tttttggtac cgggaattgg gaattgggca
     1201 atatttaaat ggcggcgcgc cgaattcttt tttgctcacc tgtgattgct cctactcaaa
     1261 tacaaaaaca tcaaattttc tgtcaataaa gcatatttat ttatatttat tttacaggaa
     1321 agaattcctt ttaaagtgta ttttaaccta taatgaaaaa cgattaaaaa aaatacataa
     1381 aataattcga aaatttttga atagcccagg ttgataaaaa ttcatttcat acgttttata
     1441 acttatgccc ctaagtattt tttgaccata gtgtttcaat tctacattaa ttttacagag
     1501 tagaatgaaa cgccacctac tcagccaaga ggcgaaaagg ttagctcgcc aagcagagag
     1561 ggcgccagtg ctcactactt tttataattc tcaacttctt tttccagact cagttcgtat
     1621 atatagacct attttcaatt taacgtcgcc tgagtgtgca tcagctgggt tttagagcta
     1681 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
     1741 gtgctttttt ttttggtacc tctagagatc cactagtgtc gacgatgtag gtcacggtct
     1801 cgaagccgcg gtgcgggtgc cagggcgtgc ccttgggctc cccgggcgcg tactccacct
     1861 cacccatctg gtccatcatg atgaacgggt cgaggtggcg gtagttgatc ccggcgaacg
     1921 cgcggcgcac cgggaagccc tcgccctcga aaccgctggg cgcggtggtc acggtgagca
     1981 cgggacgtgc gacggcgtcg gcgggtgcgg atacgcgggg cagcgtcagc gggttctcga
     2041 cggtcacggc gggcatgtcg acactagttc tagccagctt ttgttccctt tagtgagggt
     2101 taatttcgag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     2161 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     2221 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     2281 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     2341 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     2401 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     2461 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     2521 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     2581 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     2641 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     2701 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     2761 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     2821 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     2881 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     2941 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     3001 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     3061 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     3121 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     3181 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     3241 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     3301 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     3361 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     3421 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     3481 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     3541 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     3601 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     3661 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     3721 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     3781 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     3841 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     3901 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     3961 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     4021 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     4081 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     4141 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     4201 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     4261 cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     4321 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     4381 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     4441 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     4501 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     4561 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     4621 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     4681 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc
     4741 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
     4801 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
     4861 ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga
     4921 attgggtacg taccgggccc ctagtatgta tgtaagttaa taaaacccat ttttgcggaa
     4981 agtagataaa aaaaacattt ttttttttta ctgcactgga tatcattgaa cttatctgat
     5041 cagttttaaa tttacttcga tccaagggta tttgatgtac caggttcttt cgattacctc
     5101 tcactcaaaa tgacattcca ctcaaagtca gcgctgtttg cctccttctc tgtccacaga
     5161 aatatcgccg tctctttcgc cgctgcgtcc gctatctctt tcgccaccgt ttgtagcgtt
     5221 acgtagcgtc aatgtccgcc ttcagttgca ttttgtcagc ggtttcgtga cgaagctcca
     5281 agcggtttac gccatcaatt aaacacaaag tgctgtgcca aaactcctct cgcttcttat
     5341 ttttgtttgt tttttgagtg attggggtgg tgattggttt tgggtgggta agcaggggaa
     5401 agtgtgaaaa atcccggcaa tgggccaaga ggatcaggag ctattaattc gcggaggcag
     5461 caaacaccca tctgccgagc atctgaacaa tgtgagtagt acatgtgcat acatcttaag
     5521 ttcacttgat ctataggaac tgcgattgca acatcaaatt gtctgcggcg tgagaactgc
     5581 gacccacaaa aatcccaaac cgcaattgca caaacaaata gtgacacgaa acagattatt
     5641 ctggtagctg ttctcgctat ataagacaat ttttgagatc atatcatgat caagacatct
     5701 aaaggcattc attttcgact atattctttt ttacaaaaaa tataacaacc agatatttta
     5761 agctgatcct agatgcacaa aaaataaata aaagtataaa cctacttcgt aggatacttc
     5821 ggggtacttt ttgttcgggg ttagatgagc ataacgcttg tagttgatat ttgagatccc
     5881 ctatcattgc agggtgacag cggagcggct tcgcagagct gcattaacca gggcttcggg
     5941 caggccaaaa actacggcac gctccggcca cccagtccgc cggaggactc cggttcaggg
     6001 agcggccaac tagccgagaa cctcacctat gcctggcaca atatggacat ctttggggcg
     6061 gtcaatcagc cgggctccgg atggcggcag ctggtcaacc ggacacgcgg actattctgc
     6121 aacgagcgac acataccggc gcccaggaaa catttgctca agaacggtga gtttctattc
     6181 gcagtcggct gatctgtgtg aaatcttaat aaagggtcca attaccaatt tgaaactcag
     6241 tttgcggcgt ggcctatccg ggcgaacttt tggccgtgat gggcagttcc ggtgccggaa
     6301 agacgaccct gctgaatgcc cttgcctttc gatcgccgca gggcatccaa gtatcgccat
     6361 ccgggatgcg actgctcaat ggccaacctg tggacgccaa ggagatgcag gccaggtgcg
     6421 cctatgtcca gcaggatgac ctctttatcg gctccctaac ggccagggaa cacctgattt
     6481 tccaagccat ggtgcggatg ccacgacatc tgacctatcg gcagcgagtg gcccgcgtgg
     6541 atcaggtgat ccaggagctt tcgctcagca aatgtcagca cacgatcatc ggtgtgcccg
     6601 gcagggtgaa aggtctgtcc ggcggagaaa ggaagcgtct ggcattcgcc tccgaggctc
     6661 taaccgatcc gccgcttctg atctgcgatg agcccacctc cggactggac tcctttaccg
     6721 cccacagcgt cgtccaggtg ctgaagaagc tgtcgcagaa gggcaagacc gtcatcctga
     6781 ccattcatca gccgtcttcc gagctgtttg agctctttga caagatcctt ctgatggccg
     6841 agggcagggt agctttcttg ggcactccca gcgaagccgt cgacttcttt tcctagtgag
     6901 ttcgatgtgt ttattaaggg tatctagtat tacataacat ctcaactcct atccagcgtg
     6961 ggtgcccagt gtcctaccaa ctacaatccg gcggactttt acgtacaggt gttggccgtt
     7021 gtgcccggac gggagatcga gtcccgtgat cggatcgcca agatatgcga caattttgcc
     7081 attagcaaag tagcccggga tatggagcag ttgttggcca ccaaaaatct ggagaagcca
     7141 ctggagcagc cggagaatgg gtacacctac aaggccacct ggttcatgca gttccgggcg
     7201 gtcctgtggc gatcctggct gtcggtgctc aaggaaccac tcctcgtaaa agtgcgactt
     7261 attcagacaa cggtgagtgg ttccagtgga aacaaatgat ataacgctta caattcttgg
     7321 aaacaaattc gctagatttt agatagaatt gcctgattcc acacccttct tagttttttt
     7381 caatgagatg tatagtttat agttttgcag aagataaata aatttcattt aactcgcgaa
     7441 tattaatgag atgcgagtaa cattttaatt tgcagatggt tgccatcttg attggcctca
     7501 tctttttggg ccaacaactc acgcaagtgg gtgtgatgaa tatcaacgga gccatcttcc
     7561 tcttcctgac caacatgacc tttcaaaacg tctttgccac gataaatgta agtcatgttt
     7621 agaatacatt tgcatttcaa taatttacta actttctaat gaatcgattc gatttaggtg
     7681 ttcacctcag agctgccagt ttttatgagg gaggcccgaa gtcgacttta tcgctgtgac
     7741 acatactttc tgggcaaaac gattgccgaa ttgccgcttt ttctcacagt gccactggtc
     7801 ttcacggcga ttgcctatcc gatgatcgga ctgcgggccg gagtgctgca cttcttcaac
     7861 tgcctggcgc tggtcactct ggtggccaat gtgtcaacgt ccttcggata tctaatatcc
     7921 tgcgccagct cctcgacctc gatggcgctg tctgtgggtc cgccggttat cataccattc
     7981 ctgctctttg gcggcttctt cttgaactcg ggctcggtgc cagtatacct caaatggttg
     8041 tcgtacctct catggttccg ttacgccaac gagggtctgc tgattaacca atgggcggac
     8101 gtggagccgg gcgaaattag ctgcacatcg tcgaacacca cgtgccccag ttcgggcaag
     8161 gtcatcctgg agacgcttaa cttctccgcc gccgatctgc cgctggacta cgtgggtctg
     8221 gccattctca tcgtgagctt ccgggtgctc gcatatctgg ctctaagact tcgggcccga
     8281 cgcaaggagt agccgacata tatccgaaat aactgcttgt tttttttttt taccattatt
     8341 accatcgtgt ttactgttta ttgccccctc aaaaagctaa tgtaattata tttgtgccaa
     8401 taaaaacaag atatgaccta tagaatacaa gtatttcccc ttcgaacatc cccacaagta
     8461 gactttggat
//
