LOCUS Exported 6617 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION multiplex gRNA plasmid. ACCESSION . VERSION . KEYWORDS pCFD5 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6617) AUTHORS Port F, Bullock SL TITLE Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs. JOURNAL Nat Methods. 2016 Oct;13(10):852-4. doi: 10.1038/nmeth.3972. Epub 2016 Sep 5. PUBMED 27595403 REFERENCE 2 (bases 1 to 6617) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6617 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(25..42) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(196..784) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(276..295) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(955..1815) /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 1578..1597 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(1816..1920) /gene="bla" /label=AmpR promoter rep_origin 1947..2403 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2034..2053) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2244..2265 /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2529..2551 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 2543..2560 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 2544..2560 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 2570..2589 /label=T7 /note="T7 promoter, forward primer" promoter 2570..2588 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS complement(join(2780..2897,2974..3067,3159..3765,3820..3953, 4014..4174,4248..4273)) /codon_start=1 /product="tryptophan oxygenase" /label=vermilion /note="selectable marker, required to synthesize the brown eye pigment in Drosophila" /translation="MSCPYAGNGNDHDDSAVPLTTEVGKIYGEYLMLDKLLDAQCMLSE EDKRPVHDEHLFIITHQAYELWFKQIIFEFDSIRDMLDAEVIDETKTLEIVKRLNRVVL ILKLLVDQVPILETMTPLDFMDFRKYLAPASGFQSLQFRLIENKLGVLTEQRVRYNQKY SDVFSDEEARNSIRNSEKDPSLLELVQRWLERTPGLEESGFNFWAKFQESVDRFLEAQV QSAMEEPVEKAKNYRLMDIEKRREVYRSIFDPAVHDALVRRGDRRFSHRALQGAIMITF YRDEPRFSQPHQLLTLLMDIDSLITKWRYNHVIMVQRMIGSQQLGTGGSSGYQYLRSTL SDRYKVFLDLFNLSTFLIPREAIPPLDETIRKKLINKSV" protein_bind 4588..4657 /label=attB /bound_moiety="phage phi-C31 integrase" /note="attB site for the phi-C31 integrase (Groth et al., 2000)" protein_bind 4899..5284 /label=gypsy insulator /bound_moiety="Su(Hw), CP190, and Mod(mdg4)67.2" /note="chromatin insulator from Drosophila" promoter 5298..5718 /label=dU6-3 promoter /note="RNA polymerase III promoter for Drosophila U6-3 snRNA (Port et al., 2014)" misc_RNA 5824..5899 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_RNA 5986..6061 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" primer_bind complement(6372..6392) /label=T3 /note="T3 promoter, forward primer" promoter complement(6372..6390) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(6411..6427) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6411..6427) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6424..6446) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6435..6451 /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(6459..6489) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6504..6525 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." ORIGIN 1 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 61 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 121 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 181 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 241 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 301 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 361 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 421 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 481 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 541 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 601 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct 661 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 721 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 781 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 841 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 901 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 961 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 1021 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 1081 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 1141 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 1201 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 1261 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 1321 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 1381 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 1441 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 1501 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 1561 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 1621 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 1681 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 1741 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 1801 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 1861 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 1921 cacatttccc cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg 1981 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 2041 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 2101 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 2161 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 2221 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 2281 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa 2341 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa 2401 tttccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc 2461 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc 2521 agggttttcc cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc 2581 actatagggc gaattgggta caagcttgga tttattttgt tatgttatat gtattatatg 2641 tcagacataa agaaaaggaa cacatcaaat gtgataacaa agactaaaca agtaatttta 2701 ttacaccaaa acgacaaaac agtaggcaga acaaacaacg catagccaaa cattgacgaa 2761 ttggataccc tgccgattgt cagacacttt tgttgatcag tttcttgcga atggtctcgt 2821 ccagcggtgg aatcgcctcg cggggaatca gaaaagtgga cagattgaac agatccagaa 2881 acaccttgta ccgatcactg aaaccaaaaa aaaacaaagg gagaacagtt tgagttcatt 2941 gatccccgat ataatcacat ctgcgatgat cacctgagag tggagcgcag atattgatat 3001 ccagacgagc caccagtgcc caactgttgg gatccaatca tgcgttgcac catgatcacg 3061 tgattgtctg cggcgggaat agaaagtatt tggttaggaa aaccagtctt aaacataaga 3121 tatatttata aaagagtatc aaagaatgca atacttacat ctccacttgg ttattaacga 3181 gtcgatgtcc atgagcaggg tgagcaactg gtgtggttgg ctgaacctgg gttcatccct 3241 atagaaggtg atcatgatgg ctccctgaag ggcacgatgg ctaaaccggc gatccccacg 3301 acgcaccagt gcatcgtgca ctgccggatc aaagatggag cgatacacct cgcgtcgctt 3361 ctcaatgtcc atgaggcggt agtttttcgc cttctccacg ggctcctcca tggcgctctg 3421 tacctgcgcc tccaggaatc gatcgacgct ctcctgaaac ttggcccaga agttgaagcc 3481 actctcctcc agtccgggcg tcctctccag ccatcgctgc actagctcca gtagcgaggg 3541 atctttctcc gagttgcgaa tcgagttccg cgcctcctcg tcgctaaaga catccgagta 3601 cttctggttg tatctcaccc gctgctctgt cagaactccc agcttgttct cgatcaaacg 3661 gaactgcagc gactgaaaac cagatgcggg tgccaggtac ttgcggaagt ccatgaagtc 3721 tagcggggtc atggtctcca gaatgggcac ttggtccacc aggagctgta caaaggaagt 3781 tataaacgga ttttggtaag agattcagaa agcactcact tttagaatca gaaccactcg 3841 gttcagtcgc ttgacaatct ccagcgtctt ggtttcatcg atgacctctg catccaacat 3901 gtctcgtatg gagtcgaact caaagatgat ctgcttgaac caaagctcgt aggctgtggc 3961 gaaggtactt aaatgccatt gagtgttgtc atcaaagttg taaacctact caccctggtg 4021 cgtgatgatg aacagatgct catcgtgcac gggtcgcttg tcctcctcgg acagcataca 4081 ctgggcatcc agcagtttgt ccagcatcag atactctcca tagattttgc ccacttccgt 4141 ggttaatggc accgccgaat catcgtgatc gtttctgtat gggtttgaat tgaatcgcag 4201 aactgaagat cgattggcat tcctggacag cacgtgctgg tgctcacccg tttcctgcat 4261 agggacagct catggtgcac agctcagatc agatcgtgac tcctcgagcg gcggatgctg 4321 gcgaactgat ctccgccagc ggaccggaga tgagacccca gcgaaccgat aacagagcga 4381 gagagctcca gttccgactg attgcacagt cggtgatctg ggcgatgggc actgccagat 4441 aggctgggaa ttatcaatca cttgaggtga aagtgcggcg cacacaaatc caagcttgat 4501 atcatcgatc tcgaggctgc atccaacgcg ttgggagctc tccggatcaa ttcggcttca 4561 ggtaccgtcg acgatgtagg tcacggtctc gaagccgcgg tgcgggtgcc agggcgtgcc 4621 cttgggctcc ccgggcgcgt actccacctc acccatctgg tccatcatga tgaacgggtc 4681 gaggtggcgg tagttgatcc cggcgaacgc gcggcgcacc gggaagccct cgccctcgaa 4741 accgctgggc gcggtggtca cggtgagcac gggacgtgcg acggcgtcgg ctggtgcgga 4801 tacgcggggc agcgtcagcg ggttctcgac ggtcacggcg ggcatgtcga caagccgaat 4861 tgatccacta gaaggcctaa ttcggtacac tagttggcca cgtaataagt gtgcgttgaa 4921 tttattcgca aaaacattgc atattttcgg caaagtaaaa ttttgttgca taccttatca 4981 aaaaataagt gctgcatact ttttagagaa accaaataat tttttattgc atacccgttt 5041 ttaataaaat acattgcata ccctctttta ataaaaaata ttgcatactt tgacgaaaca 5101 aattttcgtt gcatacccaa taaaagatta ttatattgca tacccgtttt taataaaata 5161 cattgcatac cctcttttaa taaagaatat tgcatacgtt gacgaaacaa attttcgttg 5221 catacccaat aaaagattat tatattgcat accttttctt gccataccat ttagccgatc 5281 aattgagatc tgaattcttt tttgctcacc tgtgattgct cctactcaaa tacaaaaaca 5341 tcaaattttc tgtcaataaa gcatatttat ttatatttat tttacaggaa agaattcctt 5401 ttaaagtgta ttttaaccta taatgaaaaa cgattaaaaa aaatacataa aataattcga 5461 aaatttttga atagcccagg ttgataaaaa ttcatttcat acgttttata acttatgccc 5521 ctaagtattt tttgaccata gtgtttcaat tctacattaa ttttacagag tagaatgaaa 5581 cgccacctac tcagccaaga ggcgaaaagg ttagctcgcc aagcagagag ggcgccagtg 5641 ctcactactt tttataattc tcaacttctt tttccagact cagttcgtat atatagacct 5701 attttcaatt taacgtcggg gctttgagtg tgtgtagaca tcaagcatcg gtggttcagt 5761 ggtagaatgc tcgcctgcca cgcgggcggc ccgggttcga ttcccggccg atgcagggtc 5821 ttcgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa 5881 agtggcaccg agtcggtgct aacaaagcac cagtggtcta gtggtagaat agtaccctgc 5941 cacggtacag acccgggttc gattcccggc tggtgcagaa gacctgtttt agagctagaa 6001 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg 6061 cttttttgcc tacctggagc ctgagagttg ttcaataaaa taaaaatgtt tcgttttttt 6121 gctttcgcca gtatttatta tttttcatca atatgtattc aatttggtat gtatttagta 6181 attgtaatat atagacaatg gttttccgtt gacgtacata catctgacgt gtgtttattt 6241 agacataata gttatgtttt cacatctttt taatgttcgc ttaatgcgta tgcattctag 6301 acaattgtgc tcggcaacag tatatttgtg gtgtgccaac caacaacctg caggagctcc 6361 agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg 6421 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata 6481 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca 6541 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 6601 gcggggagag gcggttt //