LOCUS       sequence-309007-        7651 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of one of several LbCas12a crRNAs in Drosophila.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7651)
  AUTHORS   Port F, Starostecka M, Boutros M
  TITLE     Multiplexed conditional genome editing with Cas12a in Drosophila.
  JOURNAL   Proc Natl Acad Sci U S A. 2020 Aug 25. pii: 2004655117. doi: 
            10.1073/pnas.2004655117.
  PUBMED    32843348
REFERENCE   2  (bases 1 to 7651)
  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: "Proc Natl 
            Acad Sci U S A. 2020 Aug 25. pii: 2004655117. doi: 
            10.1073/pnas.2004655117."
COMMENT     Sequence Label: pCFD8
FEATURES             Location/Qualifiers
     source          1..7651
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    54..123
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        complement(342..360)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(381..397)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(381..397)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(394..416)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    405..421
                     /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(429..435,436..453,454..459))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    474..495
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(612..629)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(783..1371)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(863..882)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1542..2333,2334..2402))
                     /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     2165..2184
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2403..2507)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2533..2988)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2670..2691)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2882..2901
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3115..3137
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3129..3146
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3130..3146
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3153..3172
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3153..3171
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3205..3223)
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     CDS             join(3675..3746,4148..4421,4496..5150,5212..5527,5731..5862,
                     5933..6547)
                     /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"
     promoter        6770..7190
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
ORIGIN
        1 gtggtgtgcc aaccaacaac ggatccacta gtgtcgacga tgtaggtcac agtctcgaag
       61 ccgcggtgcg ggtgccaggg cgtgcccttg ggctccccgg gcgcgtactc cacctcaccc
      121 atctggtcca tcatgatgaa cgggtcgagg tggcggtagt tgatcccggc gaacgcgcgg
      181 cgcaccggga agccctcgcc ctcgaaaccg ctgggcgcgg tggtcacggt gagcacggga
      241 cgtgcgacgg cgtcggcggg tgcggatacg cggggcagcg tcagcgggtt ctcgacggtc
      301 acggcgggca tgtcgacact agttctagcc agcttttgtt ccctttagtg agggttaatt
      361 tcgagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
      421 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
      481 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
      541 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
      601 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
      661 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
      721 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
      781 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
      841 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
      901 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
      961 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     1021 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     1081 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     1141 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     1201 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     1261 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     1321 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     1381 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     1441 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     1501 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     1561 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     1621 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     1681 cgggacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     1741 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     1801 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     1861 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     1921 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1981 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     2041 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     2101 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     2161 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     2221 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     2281 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     2341 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     2401 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     2461 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     2521 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     2581 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     2641 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     2701 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     2761 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     2821 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     2881 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     2941 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat
     3001 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     3061 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
     3121 cacgacgttg taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg
     3181 gtacgtaccg ggcccctagt atgtatgtaa gttaataaaa cccatttttg cggaaagtag
     3241 ataaaaaaaa catttttttt ttttactgca ctggatatca ttgaacttat ctgatcagtt
     3301 ttaaatttac ttcgatccaa gggtatttga tgtaccaggt tctttcgatt acctctcact
     3361 caaaatgaca ttccactcaa agtcagcgct gtttgcctcc ttctctgtcc acagaaatat
     3421 cgccgtctct ttcgccgctg cgtccgctat ctctttcgcc accgtttgta gcgttacgta
     3481 gcgtcaatgt ccgccttcag ttgcattttg tcagcggttt cgtgacgaag ctccaagcgg
     3541 tttacgccat caattaaaca caaagtgctg tgccaaaact cctctcgctt cttatttttg
     3601 tttgtttttt gagtgattgg ggtggtgatt ggttttgggt gggtaagcag gggaaagtgt
     3661 gaaaaatccc ggcaatgggc caagaggatc aggagctatt aattcgcgga ggcagcaaac
     3721 acccatctgc cgagcatctg aacaatgtga gtagtacatg tgcatacatc ttaagttcac
     3781 ttgatctata ggaactgcga ttgcaacatc aaattgtctg cggcgtgaga actgcgaccc
     3841 acaaaaatcc caaaccgcaa ttgcacaaac aaatagtgac acgaaacaga ttattctggt
     3901 agctgttctc gctatataag acaatttttg agatcatatc atgatcaaga catctaaagg
     3961 cattcatttt cgactatatt cttttttaca aaaaatataa caaccagata ttttaagctg
     4021 atcctagatg cacaaaaaat aaataaaagt ataaacctac ttcgtaggat acttcggggt
     4081 actttttgtt cggggttaga tgagcataac gcttgtagtt gatatttgag atcccctatc
     4141 attgcagggt gacagcggag cggcttcgca gagctgcatt aaccagggct tcgggcaggc
     4201 caaaaactac ggcacgctcc ggccacccag tccgccggag gactccggtt cagggagcgg
     4261 ccaactagcc gagaacctca cctatgcctg gcacaatatg gacatctttg gggcggtcaa
     4321 tcagccgggc tccggatggc ggcagctggt caaccggaca cgcggactat tctgcaacga
     4381 gcgacacata ccggcgccca ggaaacattt gctcaagaac ggtgagtttc tattcgcagt
     4441 cggctgatct gtgtgaaatc ttaataaagg gtccaattac caatttgaaa ctcagtttgc
     4501 ggcgtggcct atccgggcga acttttggcc gtgatgggca gttccggtgc cggaaagacg
     4561 accctgctga atgcccttgc ctttcgatcg ccgcagggca tccaagtatc gccatccggg
     4621 atgcgactgc tcaatggcca acctgtggac gccaaggaga tgcaggccag gtgcgcctat
     4681 gtccagcagg atgacctctt tatcggctcc ctaacggcca gggaacacct gattttccaa
     4741 gccatggtgc ggatgccacg acatctgacc tatcggcagc gagtggcccg cgtggatcag
     4801 gtgatccagg agctttcgct cagcaaatgt cagcacacga tcatcggtgt gcccggcagg
     4861 gtgaaaggtc tgtccggcgg agaaaggaag cgtctggcat tcgcctccga ggctctaacc
     4921 gatccgccgc ttctgatctg cgatgagccc acctccggac tggactcctt taccgcccac
     4981 agcgtcgtcc aggtgctgaa gaagctgtcg cagaagggca agaccgtcat cctgaccatt
     5041 catcagccgt catccgagct gtttgagctc tttgacaaga tccttctgat ggccgagggc
     5101 agggtagctt tcttgggcac tcccagcgaa gccgtcgact tcttttccta gtgagttcga
     5161 tgtgtttatt aagggtatct agtattacat aacatctcaa ctcctatcca gcgtgggtgc
     5221 ccagtgtcct accaactaca atccggcgga cttttacgta caggtgttgg ccgttgtgcc
     5281 cggacgggag atcgagtccc gtgatcggat cgccaagata tgcgacaatt ttgccattag
     5341 caaagtagcc cgggatatgg agcagttgtt ggccaccaaa aatctggaga agccactgga
     5401 gcagccggag aatgggtaca cctacaaggc cacctggttc atgcagttcc gggcggtcct
     5461 gtggcgatcc tggctgtcgg tgctcaagga accactcctc gtaaaagtgc gacttattca
     5521 gacaacggtg agtggttcca gtggaaacaa atgatataac gcttacaatt cttggaaaca
     5581 aattcgctag attttagata gaattgcctg attccacacc cttcttagtt tttttcaatg
     5641 agatgtatag tttatagttt tgcagaagat aaataaattt catttaactc gcgaatatta
     5701 atgagatgcg agtaacattt taatttgcag atggttgcca tcttgattgg cctcatcttt
     5761 ttgggccaac aactcacgca agtgggtgtg atgaatatca acggagccat cttcctcttc
     5821 ctgaccaaca tgacctttca aaacgtcttt gccacgataa atgtaagtca tgtttagaat
     5881 acatttgcat ttcaataatt tactaacttt ctaatgaatc gattcgattt aggtgttcac
     5941 ctcagagctg ccagttttta tgagggaggc ccgaagtcga ctttatcgct gtgacacata
     6001 ctttctgggc aaaacgattg ccgaattgcc gctttttctc acagtgccac tggttttcac
     6061 ggcgattgcc tatccgatga tcggactgcg ggccggagtg ctgcacttct tcaactgcct
     6121 ggcgctggtc actctggtgg ccaatgtgtc aacgtccttc ggatatctaa tatcctgcgc
     6181 cagctcctcg acctcgatgg cgctgtctgt gggtccgccg gttatcatac cattcctgct
     6241 ctttggcggc ttcttcttga actcgggctc ggtgccagta tacctcaaat ggttgtcgta
     6301 cctctcatgg ttccgttacg ccaacgaggg tctgctgatt aaccaatggg cggacgtgga
     6361 gccgggcgaa attagctgca catcgtcgaa caccacgtgc cccagttcgg gcaaggtcat
     6421 cctggagacg cttaacttct ccgccgccga tctgccgctg gactacgtgg gtctggccat
     6481 tctcatcgtg agcttccggg tgctcgcata tctggctcta agacttcggg cccgacgcaa
     6541 ggagtagccg acatatatcc gaaataactg cttgtttttt tttttaccat tattaccatc
     6601 gtgtttactg tttattgccc cctcaaaaag ctaatgtaat tatatttgtg ccaataaaaa
     6661 caagatatga cctatagaat acaagtattt ccccttcgaa catccccaca agtagacttt
     6721 ggatttgttt ctaaccaaaa gacttacaca cctgcatacc ttagaattct tttttgctca
     6781 cctgtgattg ctcctactca aatacaaaaa catcaaattt tctgtcaata aagcatattt
     6841 atttatattt attttacagg aaagaattcc ttttaaagtg tattttaacc tataatgaaa
     6901 aacgattaaa aaaaatacat aaaataattc gaaaattttt gaatagccca ggttgataaa
     6961 aattcatttc atacgtttta taacttatgc ccctaagtat tttttgacca tagtgtttca
     7021 attctacatt aattttacag agtagaatga aacgccacct actcagccaa gaggcgaaaa
     7081 ggttagctcg ccaagcagag agggcgccag tgctcactac tttttataat tctcaacttc
     7141 tttttccaga ctcagttcgt atatatagac ctattttcaa tttaacgtcg gggctttgag
     7201 tgtgtgtaga catcaagcat cggtggttca gtggtagaat gctcgcctgc cacgcgggcg
     7261 gcccgggttc gattcccggc cgatgcataa tttctactaa gtgtagatgg gtcttcgtta
     7321 tcagaagacc taacaaagca ccagtggtct agtggtagaa tagtaccctg ccacggtaca
     7381 gacccgggtt cgattcccgg ctggtgcatt tttttgccta cctggagcct gagagttgtt
     7441 caataaaata aaaatgtttc gtttttttgc tttcgccagt atttattatt tttcatcaat
     7501 atgtattcaa tttggtatgt atttagtaat tgtaatatat agacaatggt tttccgttga
     7561 cgtacataca tctgacgtgt gtttatttag acataatagt tatgttttca catcttttta
     7621 atgttcgctt aatgcgtatg cattcgctag a
//
