LOCUS       sequence-322854-        7884 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  clone in U6 and DR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7884)
  AUTHORS   Sun R, Brogan D, Buchman A, Yang T, Akbari OS
  TITLE     Ubiquitous and Tissue-specific RNA Targeting in Drosophila 
            Melanogaster using CRISPR/CasRx.
  JOURNAL   J Vis Exp. 2021 Feb 5;(168). doi: 10.3791/62154.
  PUBMED    33616113
REFERENCE   2  (bases 1 to 7884)
  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: "J Vis 
            Exp."; date: "2021-02-5"; volume: "(168). doi"; pages: " 
            10.3791/62154"
COMMENT     Sequence Label: OA-1043
FEATURES             Location/Qualifiers
     source          1..7884
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..421
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     protein_bind    525..594
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        complement(813..831)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(852..868)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(852..868)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(865..887)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    876..892
                     /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(900..906,907..924,925..930))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    945..966
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1083..1100)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1254..1842)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1334..1353)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2013..2804,2805..2873))
                     /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     2636..2655
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2874..2978)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3004..3459)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3141..3162)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3353..3372
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3586..3608
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3600..3617
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3601..3617
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3624..3643
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3624..3642
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3676..3694)
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     CDS             join(4146..4217,4619..4892,4967..5621,5683..5998,6202..6333,
                     6404..7018)
                     /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    7793..7826
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 ttttttgctc acctgtgatt gctcctactc aaatacaaaa acatcaaatt ttctgtcaat
       61 aaagcatatt tatttatatt tattttacag gaaagaattc cttttaaagt gtattttaac
      121 ctataatgaa aaacgattaa aaaaaataca taaaataatt cgaaaatttt tgaatagccc
      181 aggttgataa aaattcattt catacgtttt ataacttatg cccctaagta ttttttgacc
      241 atagtgtttc aattctacat taattttaca gagtagaatg aaacgccacc tactcagcca
      301 agaggcgaaa aggttagctc gccaagcaga gagggcgcca gtgctcacta ctttttataa
      361 ttctcaactt ctttttccag actcagttcg tatatataga cctattttca atttaacgtc
      421 ggctgcagtt gtggaaggtc cagttttgag gggctattac aacttttttg cggccgcaac
      481 ggtacctcta gagatccact agtgtcgacg atgtaggtca cggtctcgaa gccgcggtgc
      541 gggtgccagg gcgtgccctt gggctccccg ggcgcgtact ccacctcacc catctggtcc
      601 atcatgatga acgggtcgag gtggcggtag ttgatcccgg cgaacgcgcg gcgcaccggg
      661 aagccctcgc cctcgaaacc gctgggcgcg gtggtcacgg tgagcacggg acgtgcgacg
      721 gcgtcggcgg gtgcggatac gcggggcagc gtcagcgggt tctcgacggt cacggcgggc
      781 atgtcgacac tagttctagc cagcttttgt tccctttagt gagggttaat ttcgagcttg
      841 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
      901 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
      961 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     1021 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     1081 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     1141 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     1201 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     1261 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     1321 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     1381 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     1441 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     1501 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     1561 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     1621 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     1681 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     1741 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     1801 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     1861 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     1921 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     1981 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     2041 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     2101 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     2161 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     2221 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     2281 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     2341 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     2401 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     2461 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     2521 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     2581 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     2641 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     2701 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     2761 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     2821 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     2881 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     2941 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     3001 cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     3061 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     3121 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     3181 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     3241 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     3301 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     3361 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     3421 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc
     3481 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
     3541 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
     3601 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg ggtacgtacc
     3661 gggcccctag tatgtatgta agttaataaa acccattttt gcggaaagta gataaaaaaa
     3721 acattttttt tttttactgc actggatatc attgaactta tctgatcagt tttaaattta
     3781 cttcgatcca agggtatttg atgtaccagg ttctttcgat tacctctcac tcaaaatgac
     3841 attccactca aagtcagcgc tgtttgcctc cttctctgtc cacagaaata tcgccgtctc
     3901 tttcgccgct gcgtccgcta tctctttcgc caccgtttgt agcgttacgt agcgtcaatg
     3961 tccgccttca gttgcatttt gtcagcggtt tcgtgacgaa gctccaagcg gtttacgcca
     4021 tcaattaaac acaaagtgct gtgccaaaac tcctctcgct tcttattttt gtttgttttt
     4081 tgagtgattg gggtggtgat tggttttggg tgggtaagca ggggaaagtg tgaaaaatcc
     4141 cggcaatggg ccaagaggat caggagctat taattcgcgg aggcagcaaa cacccatctg
     4201 ccgagcatct gaacaatgtg agtagtacat gtgcatacat cttaagttca cttgatctat
     4261 aggaactgcg attgcaacat caaattgtct gcggcgtgag aactgcgacc cacaaaaatc
     4321 ccaaaccgca attgcacaaa caaatagtga cacgaaacag attattctgg tagctgttct
     4381 cgctatataa gacaattttt gagatcatat catgatcaag acatctaaag gcattcattt
     4441 tcgactatat tcttttttac aaaaaatata acaaccagat attttaagct gatcctagat
     4501 gcacaaaaaa taaataaaag tataaaccta cttcgtagga tacttcgggg tactttttgt
     4561 tcggggttag atgagcataa cgcttgtagt tgatatttga gatcccctat cattgcaggg
     4621 tgacagcgga gcggcttcgc agagctgcat taaccagggc ttcgggcagg ccaaaaacta
     4681 cggcacgctc cggccaccca gtccgccgga ggactccggt tcagggagcg gccaactagc
     4741 cgagaacctc acctatgcct ggcacaatat ggacatcttt ggggcggtca atcagccggg
     4801 ctccggatgg cggcagctgg tcaaccggac acgcggacta ttctgcaacg agcgacacat
     4861 accggcgccc aggaaacatt tgctcaagaa cggtgagttt ctattcgcag tcggctgatc
     4921 tgtgtgaaat cttaataaag ggtccaatta ccaatttgaa actcagtttg cggcgtggcc
     4981 tatccgggcg aacttttggc cgtgatgggc agttccggtg ccggaaagac gaccctgctg
     5041 aatgcccttg cctttcgatc gccgcagggc atccaagtat cgccatccgg gatgcgactg
     5101 ctcaatggcc aacctgtgga cgccaaggag atgcaggcca ggtgcgccta tgtccagcag
     5161 gatgacctct ttatcggctc cctaacggcc agggaacacc tgattttcca agccatggtg
     5221 cggatgccac gacatctgac ctatcggcag cgagtggccc gcgtggatca ggtgatccag
     5281 gagctttcgc tcagcaaatg tcagcacacg atcatcggtg tgcccggcag ggtgaaaggt
     5341 ctgtccggcg gagaaaggaa gcgtctggca ttcgcctccg aggctctaac cgatccgccg
     5401 cttctgatct gcgatgagcc cacctccgga ctggactcct ttaccgccca cagcgtcgtc
     5461 caggtgctga agaagctgtc gcagaagggc aagaccgtca tcctgaccat tcatcagccg
     5521 tcttccgagc tgtttgagct ctttgacaag atccttctga tggccgaggg cagggtagct
     5581 ttcttgggca ctcccagcga agccgtcgac ttcttttcct agtgagttcg atgtgtttat
     5641 taagggtatc tagtattaca taacatctca actcctatcc agcgtgggtg cccagtgtcc
     5701 taccaactac aatccggcgg acttttacgt acaggtgttg gccgttgtgc ccggacggga
     5761 gatcgagtcc cgtgatcgga tcgccaagat atgcgacaat tttgccatta gcaaagtagc
     5821 ccgggatatg gagcagttgt tggccaccaa aaatctggag aagccactgg agcagccgga
     5881 gaatgggtac acctacaagg ccacctggtt catgcagttc cgggcggtcc tgtggcgatc
     5941 ctggctgtcg gtgctcaagg aaccactcct cgtaaaagtg cgacttattc agacaacggt
     6001 gagtggttcc agtggaaaca aatgatataa cgcttacaat tcttggaaac aaattcgcta
     6061 gattttagat agaattgcct gattccacac ccttcttagt ttttttcaat gagatgtata
     6121 gtttatagtt ttgcagaaga taaataaatt tcatttaact cgcgaatatt aatgagatgc
     6181 gagtaacatt ttaatttgca gatggttgcc atcttgattg gcctcatctt tttgggccaa
     6241 caactcacgc aagtgggtgt gatgaatatc aacggagcca tcttcctctt cctgaccaac
     6301 atgacctttc aaaacgtctt tgccacgata aatgtaagtc atgtttagaa tacatttgca
     6361 tttcaataat ttactaactt tctaatgaat cgattcgatt taggtgttca cctcagagct
     6421 gccagttttt atgagggagg cccgaagtcg actttatcgc tgtgacacat actttctggg
     6481 caaaacgatt gccgaattgc cgctttttct cacagtgcca ctggtcttca cggcgattgc
     6541 ctatccgatg atcggactgc gggccggagt gctgcacttc ttcaactgcc tggcgctggt
     6601 cactctggtg gccaatgtgt caacgtcctt cggatatcta atatcctgcg ccagctcctc
     6661 gacctcgatg gcgctgtctg tgggtccgcc ggttatcata ccattcctgc tctttggcgg
     6721 cttcttcttg aactcgggct cggtgccagt atacctcaaa tggttgtcgt acctctcatg
     6781 gttccgttac gccaacgagg gtctgctgat taaccaatgg gcggacgtgg agccgggcga
     6841 aattagctgc acatcgtcga acaccacgtg ccccagttcg ggcaaggtca tcctggagac
     6901 gcttaacttc tccgccgccg atctgccgct ggactacgtg ggtctggcca ttctcatcgt
     6961 gagcttccgg gtgctcgcat atctggctct aagacttcgg gcccgacgca aggagtagcc
     7021 gacatatatc cgaaataact gcttgttttt tttttttacc attattacca tcgtgtttac
     7081 tgtttattgc cccctcaaaa agctaatgta attatatttg tgccaataaa aacaagatat
     7141 gacctataga atacaagtat ttccccttcg aacatcccca caagtagact ttggatttgt
     7201 cttctaacca aaagacttac acacctgcat accttacatc aaaaactcgt ttatcgctac
     7261 ataaaacacc gggatatatt ttttatatac atacttttca aatcgcgcgc cctcttcata
     7321 attcacctcc accacaccac gtttcgtagt tgctctttcg ctgtctccca cccgctctcc
     7381 gcaacacatt caccttttgt tcgacgacct tggagcgact gtcgttagtt ccgcgcgatt
     7441 cggttcgctc aaatggttcc gagtggttca tttcgtctca atagaaatta gtaataaata
     7501 tttgtatgta caatttattt gctccaatat atttgtatat atttccctca cagctatatt
     7561 tattctaatt taatattatg actttttaag gtaatttttt gtgacctgtt cggagtgatt
     7621 agcgttacaa tttgaactga aagtgacatc cagtgtttgt tccttgtgta gatgcatctc
     7681 aaaaaaatgg tgggcataat agtgttgttt atatatatca aaaataacaa ctataataat
     7741 aagaatacat ttaatttaga aaatgcttgg atttcactgg aactaggcta gcataacttc
     7801 gtataatgta tgctatacga agttatgcta gcggatccgg gaattgggaa ttgggcaata
     7861 tttaaatggc ggcgcgccga attc
//
