LOCUS Exported 7578 bp ds-DNA circular SYN 10-SEP-2021 DEFINITION synthetic circular DNA. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7578) AUTHORS Enzo Mameli TITLE Direct Submission JOURNAL Exported Friday, Sep 10, 2021 from SnapGene 5.1.7 https://www.snapgene.com FEATURES Location/Qualifiers source 1..7578 /organism="synthetic DNA construct" /mol_type="other DNA" 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 presenceof 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 toinhibit 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 (Grothet al., 2000)" promoter 2319..2507 /label=U6 (CPIJ039596) /note="promoter from CPIJ039596" misc_RNA 2526..2601 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 2610..2649 /label=3' flank CPIJ039596 misc_feature 2650..2794 /label=3'end snRNAU696AB Dmel promoter 2853..5362 /label=Ac5 promoter /note="Drosophila melanogaster actin 5C promoter" CDS 5396..6112 /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 6116..6169 /codon_start=1 /product="2A peptide from Thosea asigna virus capsidprotein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 6194..6793 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" polyA_signal 6820..6941 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" protein_bind 7135..7168 /label=attB /bound_moiety="phage phi-C31 integrase" /note="minimal attB site for the phi-C31 integrase (Grothet al., 2000)" primer_bind complement(7184..7200) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" 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 acttcgaacc aaactttgta ctcctatgct 2341 aaaatgtctg cattcatggg atgtcgcgca aacgagtcca ataagaaatc aaactgctcg 2401 aattcaacgt gacgctaccc ggattgctat acccataact ttatacatcg ctagaaccgc 2461 gttcgcgctc gcttactata taagcacttt tccggcccta caaacttggg tcttcgagaa 2521 gacctgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa 2581 aaagtggcac cgagtcggtg cttttttttg tcagttcaga ttgggttagg tgaaaaggac 2641 taggggaact tttttgccta cctggagcct gagagttgtt caataaaata aaaatgtttc 2701 gtttttttgc tttcgccagt atttattatt tttcatcaat atgtattcaa tttggtatgt 2761 atttagtaat tgtaatatat agacaatggt tttcggtacc cagcatgcaa ttctatattc 2821 taaaaacaca aatgatactt ctaaaaaaaa atcatgaatg gcatcaactc tgaatcaaat 2881 ctttgcagat gcacctactt ctcatttcca ctgtcacatc atttttccag atctcgctgc 2941 ctgttatgtg gcccacaaac caagacacgt tttatggcca ttaaagctgg ctgatcgtcg 3001 ccaaacacca aatacataat gaatatgtac acattcgaga aagaagcgat caaagaagca 3061 tcttcgggcg gagtaggaga atgcggagga gaaggagaac gagctgatct agtatctctc 3121 cacaatccaa tgccaactga ccaactggcc atattcggag caatttgaag ccaatttcca 3181 tcgcctggcg atcgctccat tcttggctat atgtttttca ccgttacccg gggccatttt 3241 caaagactcg tcggcaagat aagattgtgt cactcgctgt ctctcttcat ttgtcgaaga 3301 atgctgagga atttcgcgat gacgtcggcg agtattttga agaatgagaa taatttgtat 3361 ttatacgaaa atcagttagt ggaattttct acaaaaacat gttatctata gataattttg 3421 ttgcaaaata tgttgactat gacaaagatt gtatgtatat acctttaatg tattctcatt 3481 ttcttatgta tttataatgg caatgatgat actgatgata ttttaagatg atgccagacc 3541 aaaaggcttg aatttctgcg tcttttgccg aacgcagtgc atgtgcaatt gttgtttttt 3601 ggaatattca attttcggac tgtccgcttt gatttcagtt tcttggctta ttcaaaaagc 3661 aaagtaaagc caaaaaagcg agatggcaat accaaatgcg gcaaaacggt agtggaagga 3721 aaggggtgcg gggcagcgga aggaagggtg gggcggggcg tggcggggtc tgtggctggg 3781 cgcgacgtca ccgacgttgg agccactcct ttgaccatgt gtgcgtgtgt gtattattcg 3841 tgtctcgcca ctcgccggtt gtttttttct ttttatgctg cgctctctct agcgccatct 3901 cgcttacgca tgctcaacgc accgcatgtt gccgtttcct tttatgcgtc attttggctc 3961 gaaataggca attatttaaa caaagattag tcaacgaaaa cgctaaaata aataagtcta 4021 caatatggtt acttattgcc atgtgtgtgc agccaacgat agcaacaaaa gcaacaacac 4081 aggtggcttt ccctctttca ctttttgttt gcaagccgcg tgcgagcaag acggcacgac 4141 cggcaaacgc aattacgctg acaaagagca gacgaagttt tggcgaaaaa catcaaggcg 4201 cctgatacga atgcatttgc aataacaatt gcgatattta atattgttta tgaagctgtt 4261 tgacttcaaa acacacaaaa aaaaaaataa aacaaattat ttgaaagaga attaggaatc 4321 ggacgcttat cgttagggta acaacaagaa atgcttactg agtcacagcc tctggaaaac 4381 tgccgcaagc cagagagaga gagaaaaaga gggagagcag cttagaccgc atgtgcttgt 4441 gtgtgaggcg tctctctctt cgtctctgtt gcgcaaacgc atagactgca ctgagaaaat 4501 cgattaccta ttttttatga atgaatattt gcactattac tattcaaaac tattaagata 4561 gcaatcacat tcaatagcca aatactatac cacctgagcg atgcaacgaa atgatcaatt 4621 tgagcaaaaa tgctgcatat ttaggacggc atcattatag aaatgcttct tgctgtgtac 4681 ttttctctcg tctggcagct gtttcgccgt tattgttaaa accggcttaa gttaggtgtg 4741 ttttctacga ctagtgaatg ccctactaga agatgtgtgt tgcacaaaat gtccctggaa 4801 taaccaattt gaagtgcaga tagcagtaaa cgtaagctaa tatgaatatt atttaactgt 4861 aatgttttaa tatcgctgga cattactaat aaacccacta taaacacatg tacatatgta 4921 tgttttggca tacaatgagt agttggggaa aaaatgtgta aaagcaccgt gaccatcaca 4981 gcataaagat aaccagctga agtatcgaat atgagtaacc cccaaattga atcacatgcc 5041 gcaactgata ggacccatgg aagtacactc ttcatggcga tatacaagac acacacaagc 5101 acgaacaccc agttgcggag gaaattctcc gtaaatgaaa acccaatcgg cgaacaattc 5161 atacccatat atggtaaaag ttttgaacgc gacttgagag cggagagcat tgcggctgat 5221 aaggttttag cgctaagcgg gctttataaa acgggctgcg ggaccagttt tcatatcact 5281 accgtttgag ttcttgtgct gtgtggatac tcctcccgac acaaagccgc tccatcagcc 5341 agcagtcgtc taatccagag accccggatc cagatgaatt cgcccttgtt taaacatggt 5401 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga 5461 cgtaaacggc cacaagttca gcgtgagggg cgagggcgag ggcgatgcca ccaacggcaa 5521 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt 5581 gaccaccctg agccacggcg tgcagtgctt cgcccgctac cccgaccaca tgaagcagca 5641 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa 5701 ggacgacggc acctacaaga cccgcgccga ggtgaagttc gagggcgaca ccctagtgaa 5761 ccgcatcgag ctgaagggcg tcgacttcaa ggaggacggc aacatcctgg ggcacaagct 5821 ggagtacaac ttcaacagcc acaacatcta tatcatggcc gtcaagcaga agaacggcat 5881 caaggtgaac ttcaagatcc gccacaacgt ggaggacggc agcgtgcagc tcgccgacca 5941 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca gccactacct 6001 gagcacccag tccgtgctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct 6061 ggagttccgc accgccgccg ggatcactct cggcatggac gagctgtaca agcttgaggg 6121 cagaggaagc cttctaacat gcggtgacgt ggaggagaat cccggccctg ctagcggtag 6181 cggcagcggt agcatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt 6241 cccccgggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac 6301 cgtcgacccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg 6361 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg 6421 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc 6481 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca 6541 ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg 6601 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt 6661 gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt 6721 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa 6781 gcccggtgcc tgactcgagg gatccgaatt cagacatgat aagatacatt gatgagtttg 6841 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta 6901 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc 6961 attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct 7021 acaaatgtgg tatggctgat tatgatcagt cgaccgatgc ccttgagagc cttcaaccca 7081 gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat ggtacgcgcc 7141 cggggagccc aagggcacgc cctggcaccc gaagggcgaa ttcactggcc gtcgttttac 7201 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc 7261 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc 7321 gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta 7381 tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc 7441 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 7501 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 7561 catcaccgaa acgcgcga //