LOCUS       sequence-292052-        8487 bp    DNA     circular SYN 15-APR-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 8487)
  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 8487)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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..8487
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      4..459
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(91..110)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     301..322
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     gene            666..4802
                     /gene="white"
                     /label=mini-white
                     /note="This modified version of the white gene lacks part 
                     of the first intron."
     primer_bind     complement(675..693)
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     CDS             join(1145..1216,1618..1891,1966..2620,2682..2997,3218..3349,
                     3420..4034)
                     /codon_start=1
                     /gene="white"
                     /product="Drosophila white gene eye color pigment"
                     /label=mini-white
                     /note="This modified version of the white gene lacks part 
                     of the first intron."
                     /translation="MGQEDQELLIRGGSKHPSAEHLNNGDSGAASQSCINQGFGQAKNY
                     GTLRPPSPPEDSGSGSGQLAENLTYAWHNMDIFGAVNQPGSGWRQLVNRTRGLFCNERH
                     IPAPRKHLLKNVCGVAYPGELLAVMGSSGAGKTTLLNALAFRSPQGIQVSPSGMRLLNG
                     QPVDAKEMQARCAYVQQDDLFIGSLTAREHLIFQAMVRMPRHLTYRQRVARVDQVIQEL
                     SLSKCQHTIIGVPGRVKGLSGGERKRLAFASEALTDPPLLICDEPTSGLDSFTAHSVVQ
                     VLKKLSQKGKTVILTIHQPSSELFELFDKILLMAEGRVAFLGTPSEAVDFFSYVGAQCP
                     TNYNPADFYVQVLAVVPGREIESRDRIAKICDNFAISKVARDMEQLLATKNLEKPLEQP
                     ENGYTYKATWFMQFRAVLWRSWLSVLKEPLLVKVRLIQTTMVAILIGLIFLGQQLTQVG
                     VMNINGAIFLFLTNMTFQNVFATINVFTSELPVFMREARSRLYRCDTYFLGKTIAELPL
                     FLTVPLVFTAIAYPMIGLRAGVLHFFNCLALVTLVANVSTSFGYLISCASSSTSMALSV
                     GPPVIIPFLLFGGFFLNSGSVPVYLKWLSYLSWFRYANEGLLINQWADVEPGEISCTSS
                     NTTCPSSGKVILETLNFSAADLPLDYVGLAILIVSFRVLAYLALRLRARRKE"
     protein_bind    4809..4842
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4861..5281
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        5302..5377
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        5440..5860
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        5881..5956
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    6011..6080
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        complement(6299..6317)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6338..6354)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6338..6354)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6351..6373)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6362..6378
                     /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(6386..6392,6393..6410,6411..6416))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6431..6452
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6569..6586)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6740..7328)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6820..6839)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7499..8290,8291..8359))
                     /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     8122..8141
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8360..8464)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
       61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
      181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
      241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
      301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
      361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
      421 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtacgtaccg
      661 ggcccctagt atgtatgtaa gttaataaaa cccatttttg cggaaagtag ataaaaaaaa
      721 catttttttt ttttactgca ctggatatca ttgaacttat ctgatcagtt ttaaatttac
      781 ttcgatccaa gggtatttga tgtaccaggt tctttcgatt acctctcact caaaatgaca
      841 ttccactcaa agtcagcgct gtttgcctcc ttctctgtcc acagaaatat cgccgtctct
      901 ttcgccgctg cgtccgctat ctctttcgcc accgtttgta gcgttacgta gcgtcaatgt
      961 ccgccttcag ttgcattttg tcagcggttt cgtgacgaag ctccaagcgg tttacgccat
     1021 caattaaaca caaagtgctg tgccaaaact cctctcgctt cttatttttg tttgtttttt
     1081 gagtgattgg ggtggtgatt ggttttgggt gggtaagcag gggaaagtgt gaaaaatccc
     1141 ggcaatgggc caagaggatc aggagctatt aattcgcgga ggcagcaaac acccatctgc
     1201 cgagcatctg aacaatgtga gtagtacatg tgcatacatc ttaagttcac ttgatctata
     1261 ggaactgcga ttgcaacatc aaattgtctg cggcgtgaga actgcgaccc acaaaaatcc
     1321 caaaccgcaa ttgcacaaac aaatagtgac acgaaacaga ttattctggt agctgttctc
     1381 gctatataag acaatttttg agatcatatc atgatcaaga catctaaagg cattcatttt
     1441 cgactatatt cttttttaca aaaaatataa caaccagata ttttaagctg atcctagatg
     1501 cacaaaaaat aaataaaagt ataaacctac ttcgtaggat acttcggggt actttttgtt
     1561 cggggttaga tgagcataac gcttgtagtt gatatttgag atcccctatc attgcagggt
     1621 gacagcggag cggcttcgca gagctgcatt aaccagggct tcgggcaggc caaaaactac
     1681 ggcacgctcc ggccacccag tccgccggag gactccggtt cagggagcgg ccaactagcc
     1741 gagaacctca cctatgcctg gcacaatatg gacatctttg gggcggtcaa tcagccgggc
     1801 tccggatggc ggcagctggt caaccggaca cgcggactat tctgcaacga gcgacacata
     1861 ccggcgccca ggaaacattt gctcaagaac ggtgagtttc tattcgcagt cggctgatct
     1921 gtgtgaaatc ttaataaagg gtccaattac caatttgaaa ctcagtttgc ggcgtggcct
     1981 atccgggcga acttttggcc gtgatgggca gttccggtgc cggaaagacg accctgctga
     2041 atgcccttgc ctttcgatcg ccgcagggca tccaagtatc gccatccggg atgcgactgc
     2101 tcaatggcca acctgtggac gccaaggaga tgcaggccag gtgcgcctat gtccagcagg
     2161 atgacctctt tatcggctcc ctaacggcca gggaacacct gattttccag gccatggtgc
     2221 ggatgccacg acatctgacc tatcggcagc gagtggcccg cgtggatcag gtgatccagg
     2281 agctttcgct cagcaaatgt cagcacacga tcatcggtgt gcccggcagg gtgaaaggtc
     2341 tgtccggcgg agaaaggaag cgtctggcat tcgcctccga ggcactaacc gatccgccgc
     2401 ttctgatctg cgatgagccc acctccggac tggactcatt taccgcccac agcgtcgtcc
     2461 aggtgctgaa gaagctgtcg cagaagggca agaccgtcat cctgaccatt catcagccgt
     2521 cttccgagct gtttgagctc tttgacaaga tccttctgat ggccgagggc agggtagctt
     2581 tcttgggcac tcccagcgaa gccgtcgact tcttttccta gtgagttcga tgtgtttatt
     2641 aagggtatct agcattacat tacatctcaa ctcctatcca gcgtgggtgc ccagtgtcct
     2701 accaactaca atccggcgga cttttacgta caggtgttgg ccgttgtgcc cggacgggag
     2761 atcgagtccc gtgatcggat cgccaagata tgcgacaatt ttgctattag caaagtagcc
     2821 cgggatatgg agcagttgtt ggccaccaaa aatttggaga agccactgga gcagccggag
     2881 aatgggtaca cctacaaggc cacctggttc atgcagttcc gggcggtcct gtggcgatcc
     2941 tggctgtcgg tgctcaagga accactcctc gtaaaagtgc gacttattca gacaacggtg
     3001 agtggttcca gtggaaacaa atgatataac gcttacaatt cttggaaaca aattcgctag
     3061 attttagtta gaattgcctg attccacacc cttcttagtt tttttcaatg agatgtatag
     3121 tttatagttt tgcagaaaat aaataaattt catttaactc gcgaacatgt tgaagatatg
     3181 aatattaatg agatgcgagt aacattttaa tttgcagatg gttgccatct tgattggcct
     3241 catctttttg ggccaacaac tcacgcaagt gggcgtgatg aatatcaacg gagccatctt
     3301 cctcttcctg accaacatga cctttcaaaa cgtctttgcc acgataaatg taagtcttgt
     3361 ttagaataca tttgcatatt aataatttac taactttcta atgaatcgat tcgatttagg
     3421 tgttcacctc agagctgcca gtttttatga gggaggcccg aagtcgactt tatcgctgtg
     3481 acacatactt tctgggcaaa acgattgccg aattaccgct ttttctcaca gtgccactgg
     3541 tcttcacggc gattgcctat ccgatgatcg gactgcgggc cggagtgctg cacttcttca
     3601 actgcctggc gctggtcact ctggtggcca atgtgtcaac gtccttcgga tatctaatat
     3661 cctgcgccag ctcctcgacc tcgatggcgc tgtctgtggg tccgccggtt atcataccat
     3721 tcctgctctt tggcggcttc ttcttgaact cgggctcggt gccagtatac ctcaaatggt
     3781 tgtcgtacct ctcatggttc cgttacgcca acgagggtct gctgattaac caatgggcgg
     3841 acgtggagcc gggcgaaatt agctgcacat cgtcgaacac cacgtgcccc agttcgggca
     3901 aggtcatcct ggagacgctt aacttctccg ccgccgatct gccgctggac tacgtgggtc
     3961 tggccattct catcgtgagc ttccgggtgc tcgcatatct ggctctaaga cttcgggccc
     4021 gacgcaagga gtagccgaca tatatccgaa ataactgctt gttttttttt tttaccatta
     4081 ttaccatcgt gtttactgtt tattgccccc tcaaaaagct aatgtaatta tatttgtgcc
     4141 aataaaaaca agatatgacc tatagaatac aagtatttcc ccttcgaaca tccccacaag
     4201 tagactttgg atttgtcttc taaccaaaag acttacacac ctgcatacct tacatcaaaa
     4261 actcgtttat cgctacataa aacaccggga tatatttttt atatacatac ttttcaaatc
     4321 gcgcgccctc ttcataattc acctccacca caccacgttt cgtagttgct ctttcgctgt
     4381 ctcccacccg ctctccgcaa cacattcacc ttttgttcga cgaccttgga gcgactgtcg
     4441 ttagttccgc gcgattcggt tcgctcaaat ggttccgagt ggttcatttc gtctcaatag
     4501 aaattagtaa taaatatttg tatgtacaat ttatttgctc caatatattt gtatatattt
     4561 ccctcacagc tatatttatt ctaatttaat attatgactt tttaaggtaa ttttttgtga
     4621 cctgttcgga gtgattagcg ttacaatttg aactgaaagt gacatccagt gtttgttcct
     4681 tgtgtagatg catctcaaaa aaatggtggg cataatagtg ttgtttatat atatcaaaaa
     4741 taacaactat aataataaga atacatttaa tttagaaaat gcttggattt cactggaact
     4801 aggctagcat aacttcgtat aatgtatgct atacgaagtt atgctagcgg atccgaattc
     4861 ttttttgctc acctgtgatt gctcctactc aaatacaaaa acatcaaatt ttctgtcaat
     4921 aaagcatatt tatttatatt tattttacag gaaagaattc cttttaaagt gtattttaac
     4981 ctataatgaa aaacgattaa aaaaaataca taaaataatt cgaaaatttt tgaatagccc
     5041 aggttgataa aaattcattt catacgtttt ataacttatg cccctaagta ttttttgacc
     5101 atagtgtttc aattctacat taattttaca gagtagaatg aaacgccacc tactcagcca
     5161 agaggcgaaa aggttagctc gccaagcaga gagggcgcca gtgctcacta ctttttataa
     5221 ttctcaactt ctttttccag actcagttcg tatatataga cctattttca atttaacgtc
     5281 ggattccgta ctttgcagac ggttttagag ctagaaatag caagttaaaa taaggctagt
     5341 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttttttggt accgggaatt
     5401 gggaattggg caatatttaa atggcggcgc gccgaattct tttttgctca cctgtgattg
     5461 ctcctactca aatacaaaaa catcaaattt tctgtcaata aagcatattt atttatattt
     5521 attttacagg aaagaattcc ttttaaagtg tattttaacc tataatgaaa aacgattaaa
     5581 aaaaatacat aaaataattc gaaaattttt gaatagccca ggttgataaa aattcatttc
     5641 atacgtttta taacttatgc ccctaagtat tttttgacca tagtgtttca attctacatt
     5701 aattttacag agtagaatga aacgccacct actcagccaa gaggcgaaaa ggttagctcg
     5761 ccaagcagag agggcgccag tgctcactac tttttataat tctcaacttc tttttccaga
     5821 ctcagttcgt atatatagac ctattttcaa tttaacgtcg cctgagtgtg catcagctgg
     5881 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     5941 ggcaccgagt cggtgctttt ttttttggta cctctagaga tccactagtg tcgacgatgt
     6001 aggtcacggt ctcgaagccg cggtgcgggt gccagggcgt gcccttgggc tccccgggcg
     6061 cgtactccac ctcacccatc tggtccatca tgatgaacgg gtcgaggtgg cggtagttga
     6121 tcccggcgaa cgcgcggcgc accgggaagc cctcgccctc gaaaccgctg ggcgcggtgg
     6181 tcacggtgag cacgggacgt gcgacggcgt cggcgggtgc ggatacgcgg ggcagcgtca
     6241 gcgggttctc gacggtcacg gcgggcatgt cgacactagt tctagccagc ttttgttccc
     6301 tttagtgagg gttaatttcg agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     6361 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     6421 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     6481 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     6541 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     6601 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     6661 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     6721 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     6781 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     6841 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     6901 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     6961 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     7021 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     7081 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     7141 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     7201 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     7261 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     7321 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     7381 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     7441 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     7501 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     7561 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     7621 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     7681 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     7741 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     7801 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     7861 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     7921 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     7981 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     8041 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     8101 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     8161 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     8221 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     8281 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     8341 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     8401 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     8461 cgcgcacatt tccccgaaaa gtgccac
//
