LOCUS Exported 7731 bp DNA circular SYN 24-JAN-2022 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7731) AUTHORS . TITLE Direct Submission JOURNAL Exported Feb 8, 2022 from SnapGene 5.3.2 https://www.snapgene.com FEATURES Location/Qualifiers source 1..7731 /organism="synthetic DNA construct" /mol_type="other DNA" source 2317..2944 /label=pCFD3 /note="pCFD3" source 2953..5523 /note="/dnas_title=pLib11.6" source 5538..7284 /label=pAc5-STABLE1 /note="pAc5-STABLE1" promoter 96..200 /gene="bla" /label=AmpR promoter CDS 201..1061 /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" rep_origin 1232..1820 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" protein_bind 2108..2129 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 2144..2174 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2182..2198 /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)." primer_bind 2206..2222 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 2276..2309 /label=attB /bound_moiety="phage phi-C31 integrase" /note="minimal attB site for the phi-C31 integrase (Groth et al., 2000)" promoter 2317..2575 /label=Drosophila U6:3 promoter /note="Drosophila U6:3 promoter" misc_feature 2576..2605 /label=RfxCas13d DR30 /note="RfxCas13d DR30" misc_feature 2608..2613 /label=Left-facing BbsI site /note="Left-facing BbsI site" misc_feature 2616..2621 /label=Right-facing BbsI site /note="Right-facing BbsI site" misc_feature 2624..2630 /label=U6 terminator /note="U6 terminator" promoter 3003..5512 /label=Ac5 promoter /note="Drosophila melanogaster actin 5C promoter" CDS 5546..6262 /codon_start=1 /product="enhanced blue variant of GFP (Ai et al., 2007)" /label=EBFP2 /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL EFRTAAGITLGMDELYK" CDS 6269..6322 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 6347..6946 /codon_start=1 /gene="pac from Streptomyces" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVECPKDRATWCMTRKPGA" polyA_signal 6973..7094 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" protein_bind 7288..7321 /label=attB /bound_moiety="phage phi-C31 integrase" /note="minimal attB site for the phi-C31 integrase (Groth et al., 2000)" ORIGIN 1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2221 accatgatta cgccaagctt gcatgcctgc aggtcgactc tagaggatcc cccgggtgcc 2281 agggcgtgcc cttgggctcc ccgggcgcgt acttcgaaaa tttttgaata gcccaggttg 2341 ataaaaattc atttcatacg ttttataact tatgccccta agtatttttt gaccatagtg 2401 tttcaattct acattaattt tacagagtag aatgaaacgc cacctactca gccaagaggc 2461 gaaaaggtta gctcgccaag cagagagggc gccagtgctc actacttttt ataattctca 2521 acttcttttt ccagactcag ttcgtatata tagacctatt ttcaatttaa cgtcgaaccc 2581 ctaccaactg gtcggggttt gaaacgggtc ttcgagaaga cctttttttt ggtaccgttt 2641 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca 2701 ccgagtcggt gcttttttgc ctacctggag cctgagagtt gttcaataaa ataaaaatgt 2761 ttcgtttttt tgctttcgcc agtatttatt atttttcatc aatatgtatt caatttggta 2821 tgtatttagt aattgtaata tatagacaat ggttttccgt tgacgtacat acatctgacg 2881 tgtgtttatt tagacataat agttatgttt tcacatcttt ttaatgttcg cttaatgcgt 2941 atgcggtacc cagcatgcaa ttctatattc taaaaacaca aatgatactt ctaaaaaaaa 3001 atcatgaatg gcatcaactc tgaatcaaat ctttgcagat gcacctactt ctcatttcca 3061 ctgtcacatc atttttccag atctcgctgc ctgttatgtg gcccacaaac caagacacgt 3121 tttatggcca ttaaagctgg ctgatcgtcg ccaaacacca aatacataat gaatatgtac 3181 acattcgaga aagaagcgat caaagaagca tcttcgggcg gagtaggaga atgcggagga 3241 gaaggagaac gagctgatct agtatctctc cacaatccaa tgccaactga ccaactggcc 3301 atattcggag caatttgaag ccaatttcca tcgcctggcg atcgctccat tcttggctat 3361 atgtttttca ccgttacccg gggccatttt caaagactcg tcggcaagat aagattgtgt 3421 cactcgctgt ctctcttcat ttgtcgaaga atgctgagga atttcgcgat gacgtcggcg 3481 agtattttga agaatgagaa taatttgtat ttatacgaaa atcagttagt ggaattttct 3541 acaaaaacat gttatctata gataattttg ttgcaaaata tgttgactat gacaaagatt 3601 gtatgtatat acctttaatg tattctcatt ttcttatgta tttataatgg caatgatgat 3661 actgatgata ttttaagatg atgccagacc aaaaggcttg aatttctgcg tcttttgccg 3721 aacgcagtgc atgtgcaatt gttgtttttt ggaatattca attttcggac tgtccgcttt 3781 gatttcagtt tcttggctta ttcaaaaagc aaagtaaagc caaaaaagcg agatggcaat 3841 accaaatgcg gcaaaacggt agtggaagga aaggggtgcg gggcagcgga aggaagggtg 3901 gggcggggcg tggcggggtc tgtggctggg cgcgacgtca ccgacgttgg agccactcct 3961 ttgaccatgt gtgcgtgtgt gtattattcg tgtctcgcca ctcgccggtt gtttttttct 4021 ttttatgctg cgctctctct agcgccatct cgcttacgca tgctcaacgc accgcatgtt 4081 gccgtttcct tttatgcgtc attttggctc gaaataggca attatttaaa caaagattag 4141 tcaacgaaaa cgctaaaata aataagtcta caatatggtt acttattgcc atgtgtgtgc 4201 agccaacgat agcaacaaaa gcaacaacac aggtggcttt ccctctttca ctttttgttt 4261 gcaagccgcg tgcgagcaag acggcacgac cggcaaacgc aattacgctg acaaagagca 4321 gacgaagttt tggcgaaaaa catcaaggcg cctgatacga atgcatttgc aataacaatt 4381 gcgatattta atattgttta tgaagctgtt tgacttcaaa acacacaaaa aaaaaaataa 4441 aacaaattat ttgaaagaga attaggaatc ggacgcttat cgttagggta acaacaagaa 4501 atgcttactg agtcacagcc tctggaaaac tgccgcaagc cagagagaga gagaaaaaga 4561 gggagagcag cttagaccgc atgtgcttgt gtgtgaggcg tctctctctt cgtctctgtt 4621 gcgcaaacgc atagactgca ctgagaaaat cgattaccta ttttttatga atgaatattt 4681 gcactattac tattcaaaac tattaagata gcaatcacat tcaatagcca aatactatac 4741 cacctgagcg atgcaacgaa atgatcaatt tgagcaaaaa tgctgcatat ttaggacggc 4801 atcattatag aaatgcttct tgctgtgtac ttttctctcg tctggcagct gtttcgccgt 4861 tattgttaaa accggcttaa gttaggtgtg ttttctacga ctagtgaatg ccctactaga 4921 agatgtgtgt tgcacaaaat gtccctggaa taaccaattt gaagtgcaga tagcagtaaa 4981 cgtaagctaa tatgaatatt atttaactgt aatgttttaa tatcgctgga cattactaat 5041 aaacccacta taaacacatg tacatatgta tgttttggca tacaatgagt agttggggaa 5101 aaaatgtgta aaagcaccgt gaccatcaca gcataaagat aaccagctga agtatcgaat 5161 atgagtaacc cccaaattga atcacatgcc gcaactgata ggacccatgg aagtacactc 5221 ttcatggcga tatacaagac acacacaagc acgaacaccc agttgcggag gaaattctcc 5281 gtaaatgaaa acccaatcgg cgaacaattc atacccatat atggtaaaag ttttgaacgc 5341 gacttgagag cggagagcat tgcggctgat aaggttttag cgctaagcgg gctttataaa 5401 acgggctgcg ggaccagttt tcatatcact accgtttgag ttcttgtgct gtgtggatac 5461 tcctcccgac acaaagccgc tccatcagcc agcagtcgtc taatccagag accccggatc 5521 cagatgaatt cgcccttgtt taaacatggt gagcaagggc gaggagctgt tcaccggggt 5581 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgagggg 5641 cgagggcgag ggcgatgcca ccaacggcaa gctgaccctg aagttcatct gcaccaccgg 5701 caagctgccc gtgccctggc ccaccctcgt gaccaccctg agccacggcg tgcagtgctt 5761 cgcccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg 5821 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc acctacaaga cccgcgccga 5881 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggcg tcgacttcaa 5941 ggaggacggc aacatcctgg ggcacaagct ggagtacaac ttcaacagcc acaacatcta 6001 tatcatggcc gtcaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacgt 6061 ggaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg 6121 ccccgtgctg ctgcccgaca gccactacct gagcacccag tccgtgctga gcaaagaccc 6181 caacgagaag cgcgatcaca tggtcctgct ggagttccgc accgccgccg ggatcactct 6241 cggcatggac gagctgtaca agaagcttga gggcagagga agccttctaa catgcggtga 6301 cgtggaggag aatcccggcc ctgctagcgg tagcggcagc ggtagcatga ccgagtacaa 6361 gcccacggtg cgcctcgcca cccgcgacga cgtcccccgg gccgtacgca ccctcgccgc 6421 cgcgttcgcc gactaccccg ccacgcgcca caccgtcgac ccggaccgcc acatcgagcg 6481 ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg 6541 ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg 6601 ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc 6661 gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct 6721 ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct 6781 ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc 6841 ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgagtgccc 6901 gaaggaccgc gcgacctggt gcatgacccg caagcccggt gcctgactcg agggatccga 6961 attcagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa 7021 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct 7081 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg 7141 tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggct gattatgatc 7201 agtcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc 7261 atgactatcg tcgccgcact tatggtacgc gcccggggag cccaagggca cgccctggca 7321 cccgaagggc gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 7381 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 7441 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga 7501 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca 7561 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg 7621 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct 7681 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg a //