LOCUS Exported 4794 bp ds-DNA circular SYN 11-DEC-2018 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4794) AUTHORS Molecular Function Laboratory TITLE Direct Submission JOURNAL Exported Dec 11, 2018 from SnapGene Viewer 4.2.9 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4794 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 4..459 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 600..616 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 626..644 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" misc_feature 701..706 /label=kozak seq CDS 707..1729 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" primer_bind 1587..1609 /label=(1241) Hyg seq1 CDS 1742..1798 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 1805..2009 /codon_start=1 /label=Linker-mAID-Clover /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA AAFVKVSMDGAPYLRKIDLRMYK" CDS 1805..2008 /codon_start=1 /label=mAID /note="note=""direct strand translation""translation=""KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNV MVSCQKSSGGPEAAAFVKVSMDGAPYLRKIDLRMYK""" /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA AAFVKVSMDGAPYLRKIDLRMYK" CDS 2015..2527 /codon_start=1 /product="NanoLuc(R) luciferase" /label=Nluc /note="human codon-optimized" /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW RLCERILA" primer_bind complement(2503..2551) /label=(1843) Nluc-NarI CDS 2528..2551 /codon_start=1 /label=Linker /translation="GAGAGAGA" CDS 2548..2551 /codon_start=1 /label=Linker-mAID-Clover /translation="A" misc_feature 2548..2549 /label=(965)_pAID_Linker_5'_BamHI_SacI promoter complement(2606..2624) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(2641..2659) /label=(2) M13 reverse primer primer_bind complement(2645..2661) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 2669..2685 /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(2693..2723) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2738..2759 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(3047..3635) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(3806..4666) /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" promoter complement(4667..4771) /gene="bla" /label=AmpR promoter ORIGIN 1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg 241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt 361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat 421 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg 481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg 661 gccccccctc gaggtcgacg gtatcgataa gcttgatatc gccaccatga aaaagcctga 721 actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct 781 gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg 841 atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg 901 gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga 961 gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga 1021 aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc 1081 cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac 1141 tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt 1201 gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc 1261 cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct 1321 gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc 1381 ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca 1441 gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta 1501 tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga 1561 tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg 1621 gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact 1681 cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat caggaagcgg 1741 agctactaac ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc ctggacctag 1801 atctaaggag aagagtgctt gtcctaaaga tccagccaaa cctccggcca aggcacaagt 1861 tgtgggatgg ccaccggtga gatcataccg gaagaacgtg atggtttcct gccaaaaatc 1921 aagcggtggc ccggaggcgg cggcgttcgt gaaggtatca atggacggag caccgtactt 1981 gaggaaaatc gatttgagga tgtataaagc tagcatggtc ttcacactcg aagatttcgt 2041 tggggactgg cgacagacag ccggctacaa cctggaccaa gtccttgaac agggaggtgt 2101 gtccagtttg tttcagaatc tcggggtgtc cgtaactccg atccaaagga ttgtcctgag 2161 cggtgaaaat gggctgaaga tcgacatcca tgtcatcatc ccgtatgaag gtctgagcgg 2221 cgaccaaatg ggccagatcg aaaaaatttt taaggtggtg taccctgtgg atgatcatca 2281 ctttaaggtg atcctgcact atggcacact ggtaatcgac ggggttacgc cgaacatgat 2341 cgactatttc ggacggccgt atgaaggcat cgccgtgttc gacggcaaaa agatcactgt 2401 aacagggacc ctgtggaacg gcaacaaaat tatcgacgag cgcctgatca accccgacgg 2461 ctccctgctg ttccgagtaa ccatcaacgg agtgaccggc tggcggctgt gcgaacgcat 2521 tctggcgggc gctggtgcag gggccggcgc cggatccact agttctagag cggccgccac 2581 cgcggtggag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat 2641 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac 2701 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 2761 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 2821 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 2881 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 2941 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 3001 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 3061 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 3121 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 3181 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 3241 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 3301 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 3361 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 3421 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 3481 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 3541 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 3601 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 3661 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 3721 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 3781 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 3841 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 3901 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 3961 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 4021 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 4081 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 4141 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 4201 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 4261 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 4321 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 4381 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 4441 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 4501 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 4561 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 4621 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 4681 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 4741 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccac //