LOCUS Exported 6928 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Arabinose inducible lambda red and anhydrotetracycline inducible sgRNA expression. ACCESSION . VERSION . KEYWORDS pKDsgRNA-ack SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6928) AUTHORS Reisch CR, Prather KL TITLE The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli. JOURNAL Sci Rep. 2015 Oct 14;5:15096. doi: 10.1038/srep15096. PUBMED 26463009 REFERENCE 2 (bases 1 to 6928) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6928 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(11..889) /codon_start=1 /gene="araC" /product="L-arabinose regulatory protein" /label=araC /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE EKVNDVAVKLS" promoter 916..1200 /gene="araBAD" /label=araBAD promoter /note="promoter of the L-arabinose operon of E. coli; the araC regulatory gene is transcribed in the opposite direction (Guzman et al., 1995)" primer_bind 1123..1142 /label=pBAD Forward /note="For vectors with E. coli araBAD promoter, forward primer" RBS 1227..1235 /label=Shine-Dalgarno sequence /note="full consensus sequence for ribosome-binding sites upstream of start codons in E. coli; complementary to a region in the 3' end of the 16S rRNA (Chen et al., 1994)" CDS 1244..1660 /codon_start=1 /gene="gam" /product="inhibitor of the host RecBCD nuclease in the lambda Red system" /label=Gam /note="protects the double-stranded DNA substrate for recombination" /translation="MDINTETEIKQKHSLTPFPVFLISPAFRGRYFHSYFRSSAMNAYY IQDRLEAQSWARHYQQLAREEKEAELADDMEKGLPQHLFESLCIDHLQRHGASKKSITR AFDDDVEFQERMAEHIRYMVETIAHHQVDIDSEV" CDS 1666..2451 /codon_start=1 /gene="bet" /product="single-stranded DNA binding recombinase in the lambda Red system" /label=Beta /note="mediates pairing of a single-stranded DNA with a complementary target" /translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLA FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA" CDS 2448..3128 /codon_start=1 /gene="exo" /product="5' to 3' double-stranded DNA exonuclease in the lambda Red system" /label=Exo /note="creates 3' single-stranded DNA tails" /translation="MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKPR SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG EQWR" terminator 3129..3373 /label=-lambda tL3 terminator /note="transcription terminator tL3 from phage lambda" CDS complement(3496..4446) /codon_start=1 /gene="rep101 encoding an A56V mutation" /product="temperature-sensitive version of the RepA protein needed for replication with the pSC101 origin (Armstrong et al., 1984)" /label=Rep101(Ts) /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI" rep_origin 4494..4716 /label=pSC101 ori /note="low-copy replication origin that requires the Rep101 protein" protein_bind 5282..5300 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 5307..5325 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" misc_RNA 5356..5431 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" CDS 5452..5481 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 5497..5514 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(5570..5587) /label=pBAD Reverse /note="For vectors with E. coli araBAD promoter, reverse primer" terminator 5740..5786 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" CDS complement(5925..6716) /codon_start=1 /gene="aadA" /product="aminoglycoside adenylyltransferase (Murphy, 1985)" /label=SmR /note="confers resistance to spectinomycin and streptomycin" /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK" promoter complement(6717..6808) /gene="bla" /label=AmpR promoter ORIGIN 1 catcgattta ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac 61 ggaactcgct cgggctggcc ccggtgcatt ttttaaatac ccgcgagaag tagagttgat 121 cgtcaaaacc aacattgcga ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca 181 gcttcgcctg gctgatacgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct 241 ggcggaaaag atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga 301 tatcaaaatt gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat 361 tatccatcgg tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct 421 caagcagatt tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga 481 tttgcccaaa caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaccccg 541 tattggcaaa tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt 601 aaacccactg gtgataccat tcgcgagcct ccggatgacg accgtagtga tgaatctctc 661 ctggcgggaa cagcaaaata tcacccggtc ggcaaacaaa ttctcgtccc tgatttttca 721 ccaccccctg accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt 781 cgataaaaaa atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg 841 cattaaacga gtatcccggc agcaggggat cattttgcgc ttcagccata cttttcatac 901 tcccgccatt cagagaagaa accaattgtc catattgcat cagacattgc cgtcactgcg 961 tcttttactg gctcttctcg ctaaccaaac cggtaacccc gcttattaaa agcattctgt 1021 aacaaagcgg gaccaaagcc atgacaaaaa cgcgtaacaa aagtgtctat aatcacggca 1081 gaaaagtcca cattgattat ttgcacggcg tcacactttg ctatgccata gcatttttat 1141 ccataagatt agcggatcct acctgacgct ttttatcgca actctctact gtttctccat 1201 acccgttttt ttgggaattc gagctctaag gaggttataa aaaatggata ttaatactga 1261 aactgagatc aagcaaaagc attcactaac cccctttcct gttttcctaa tcagcccggc 1321 atttcgcggg cgatattttc acagctattt caggagttca gccatgaacg cttattacat 1381 tcaggatcgt cttgaggctc agagctgggc gcgtcactac cagcagctcg cccgtgaaga 1441 gaaagaggca gaactggcag acgacatgga aaaaggcctg ccccagcacc tgtttgaatc 1501 gctatgcatc gatcatttgc aacgccacgg ggccagcaaa aaatccatta cccgtgcgtt 1561 tgatgacgat gttgagtttc aggagcgcat ggcagaacac atccggtaca tggttgaaac 1621 cattgctcac caccaggttg atattgattc agaggtataa aacgaatgag tactgcactc 1681 gcaacgctgg ctgggaagct ggctgaacgt gtcggcatgg attctgtcga cccacaggaa 1741 ctgatcacca ctcttcgcca gacggcattt aaaggtgatg ccagcgatgc gcagttcatc 1801 gcattactga tcgttgccaa ccagtacggc cttaatccgt ggacgaaaga aatttacgcc 1861 tttcctgata agcagaatgg catcgttccg gtggtgggcg ttgatggctg gtcccgcatc 1921 atcaatgaaa accagcagtt tgatggcatg gactttgagc aggacaatga atcctgtaca 1981 tgccggattt accgcaagga ccgtaatcat ccgatctgcg ttaccgaatg gatggatgaa 2041 tgccgccgcg aaccattcaa aactcgcgaa ggcagagaaa tcacggggcc gtggcagtcg 2101 catcccaaac ggatgttacg tcataaagcc atgattcagt gtgcccgtct ggccttcgga 2161 tttgctggta tctatgacaa ggatgaagcc gagcgcattg tcgaaaatac tgcatacact 2221 gcagaacgtc agccggaacg cgacatcact ccggttaacg atgaaaccat gcaggagatt 2281 aacactctgc tgatcgccct ggataaaaca tgggatgacg acttattgcc gctctgttcc 2341 cagatatttc gccgcgacat tcgtgcatcg tcagaactga cacaggccga agcagtaaaa 2401 gctcttggat tcctgaaaca gaaagccgca gagcagaagg tggcagcatg acaccggaca 2461 ttatcctgca gcgtaccggg atcgatgtga gagctgtcga acagggggat gatgcgtggc 2521 acaaattacg gctcggcgtc atcaccgctt cagaagttca caacgtgata gcaaaacccc 2581 gctccggaaa gaagtggcct gacatgaaaa tgtcctactt ccacaccctg cttgctgagg 2641 tttgcaccgg tgtggctccg gaagttaacg ctaaagcact ggcctgggga aaacagtacg 2701 agaacgacgc cagaaccctg tttgaattca cttccggcgt gaatgttact gaatccccga 2761 tcatctatcg cgacgaaagt atgcgtaccg cctgctctcc cgatggttta tgcagtgacg 2821 gcaacggcct tgaactgaaa tgcccgttta cctcccggga tttcatgaag ttccggctcg 2881 gtggtttcga ggccataaag tcagcttaca tggcccaggt gcagtacagc atgtgggtga 2941 cgcgaaaaaa tgcctggtac tttgccaact atgacccgcg tatgaagcgt gaaggcctgc 3001 attatgtcgt gattgagcgg gatgaaaagt acatggcgag ttttgacgag atcgtgccgg 3061 agttcatcga aaaaatggac gaggcactgg ctgaaattgg ttttgtattt ggggagcaat 3121 ggcgatgacg catcctcacg ataatatccg ggtaggcgca atcactttcg tctactccgt 3181 tacaaagcga ggctgggtat ttcccggcct ttctgttatc cgaaatccac tgaaagcaca 3241 gcggctggct gaggagataa ataataaacg aggggctgta tgcacaaagc atcttctgtt 3301 gagttaagaa cgagtatcga gatggcacat agccttgctc aaattggaat caggtttgtg 3361 ccaataccag tagaaacaga cgaagaatcc atgggtatgg acagttttcc ctttgatatg 3421 taacggtgaa cagttgttct acttttgttt gttagtcttg atgcttcact gatagataca 3481 agagccataa gaacctcaga tccttccgta tttagccagt atgttctcta gtgtggttcg 3541 ttgtttttgc gtgagccatg agaacgaacc attgagatca tacttacttt gcatgtcact 3601 caaaaatttt gcctcaaaac tggtgagctg aatttttgca gttaaagcat cgtgtagtgt 3661 ttttcttagt ccgttatgta ggtaggaatc tgatgtaatg gttgttggta ttttgtcacc 3721 attcattttt atctggttgt tctcaagttc ggttacgaga tccatttgtc tatctagttc 3781 aacttggaaa atcaacgtat cagtcgggcg gcctcgctta tcaaccacca atttcatatt 3841 gctgtaagtg tttaaatctt tacttattgg tttcaaaacc cattggttaa gccttttaaa 3901 ctcatggtag ttattttcaa gcattaacat gaacttaaat tcatcaaggc taatctctat 3961 atttgccttg tgagttttct tttgtgttag ttcttttaat aaccactcat aaatcctcat 4021 agagtatttg ttttcaaaag acttaacatg ttccagatta tattttatga atttttttaa 4081 ctggaaaaga taaggcaata tctcttcact aaaaactaat tctaattttt cgcttgagaa 4141 cttggcatag tttgtccact ggaaaatctc aaagccttta accaaaggat tcctgatttc 4201 cacagttctc gtcatcagct ctctggttgc tttagctaat acaccataag cattttccct 4261 actgatgttc atcatctgaa cgtattggtt ataagtgaac gataccgtcc gttctttcct 4321 tgtagggttt tcaatcgtgg ggttgagtag tgccacacag cataaaatta gcttggtttc 4381 atgctccgtt aagtcatagc gactaatcgc tagttcattt gctttgaaaa caactaattc 4441 agacatacat ctcaattggt ctaggtgatt ttaatcacta taccaattga gatgggctag 4501 tcaatgataa ttactagtcc ttttcctttg agttgtgggt atctgtaaat tctgctagac 4561 ctttgctgga aaacttgtaa attctgctag accctctgta aattccgcta gacctttgtg 4621 tgtttttttt gtttatattc aagtggttat aatttataga ataaagaaag aataaaaaaa 4681 gataaaaaga atagatccca gccctgtgta taactcacta ctttagtcag ttccgcagta 4741 ttacaaaagg atgtcgcaaa cgctgtttgc tcctctacaa aacagacctt aaaaccctaa 4801 aggcttaagt agcaccctcg caagctcggt tgcggccgca atcgggcaaa tcgctgaata 4861 ttccttttgt ctccgaccat caggcacctg agtcgctgtc tttttcgtga cattcagttc 4921 gctgcgctca cggctctggc agtgaatggg ggtaaatggc actacaggcg ccttttatgg 4981 attcatgcaa ggaaactacc cataatacaa gaaaagcccg tcacgggctt ctcagggcgt 5041 tttatggcgg gtctgctatg tggtgctatc tgactttttg ctgttcagca gttcctgccc 5101 tctgattttc cagtctgacc acttcggatt atcccgtgac aggtcattca gactggctaa 5161 tgcacccagt aaggcagcgg tatcatcaac ggggtctgac gctcagtgga acgaaaactc 5221 acgttaaggg attttggtca tgagattatc agctttcgct aaggatgatt tctggaattc 5281 ttccctatca gtgatagaga ttgacatccc tatcagtgat agagatactg agcacaataa 5341 acaggaagcg gctttgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta 5401 tcaacttgaa aaagtggcac cgagtcggtg ctttttttga agcttgggcc cgaacaaaaa 5461 ctcatctcag aagaggatct gaatagcgcc gtcgaccatc atcatcatca tcattgagtt 5521 taaacggtct ccagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga 5581 ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg 5641 tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg 5701 tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag 5761 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaactggat ccttactcga 5821 gtctagactg caggcggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 5881 caatctaaag tatatatgag taaacttggt ctgctgacag gacattattt gccgactacc 5941 ttggtgatct cgcctttcac gtagtggaca aattcttcca actgatctgc gcgcgaggcc 6001 aagcgatctt cttcttgtcc aagataagcc tgtctagctt caagtatgac gggctgatac 6061 tgggccggca ggcgctccat tgcccagtcg gcagcgacat ccttcggcgc gattttgccg 6121 gttactgcgc tgtaccaaat gcgggacaac gtaagcacta catttcgctc atcgccagcc 6181 cagtcgggcg gcgagttcca tagcgttaag gtttcattta gcgcctcaaa tagatcctgt 6241 tcaggaaccg gatcaaagag ttcctccgcc gctggaccta ccaaggcaac gctatgttct 6301 cttgcttttg tcagcaagat agccagatca atgtcgatcg tggctggctc gaagatacct 6361 gcaagaatgt cattgcgctg ccattctcca aattgcagtt cgcgcttagc tggataacgc 6421 cacggaatga tgtcgtcgtg cacaacaatg gtgacttcta cagcgcggag aatctcgctc 6481 tctccagggg aagccgaagt ttccaaaagg tcgttgatca aagctcgccg cgttgtttca 6541 tcaagcctta cggtcaccgt aaccagcaaa tcaatatcac tgtgtggctt caggccgcca 6601 tccactgcgg agccgtacaa atgtacggcc agcaacgtcg gttcgagatg gcgctcgatg 6661 acgccaacta cctctgatag ttgagtcgat acttcggcga tcaccgcttc cctcatactc 6721 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 6781 tttgaatgta tttagaaaaa taaacaaata gctagctcac tcggtcgcta ccagggttat 6841 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 6901 cgcacatttc cccgaaaagt gccacctg //