LOCUS Exported 3581 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION For cloning and constitutive expression of sgRNAs in E.coli. New sgRNAs are cloned into SapI sites and multiplex sgRNAs are cloned into BsaI sites. Kanamycin resistant with pSC101 origin of rep.. ACCESSION . VERSION . KEYWORDS pSG4K5 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3581) AUTHORS Standage-Beier K, Zhang Q, Wang X TITLE Targeted Large-Scale Deletion of Bacterial Genomes Using CRISPR-Nickases. JOURNAL ACS Synth Biol. 2015 Nov 20;4(11):1217-25. doi: 10.1021/acssynbio.5b00132. Epub 2015 Oct 25. PUBMED 26451892 REFERENCE 2 (bases 1 to 3581) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3581 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 8..42 /label=J23119 promoter /note="bacterial promoter (Registry of Standard Biological Parts BBa_J23119)" misc_RNA 65..140 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" terminator 241..272 /label=tonB terminator /note="bidirectional E. coli tonB-P14 transcription terminator" CDS complement(371..1186) /codon_start=1 /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 1094..1113 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 1178..1197 /label=pENTR-R /note="pENTR vectors, reverse primer" CDS complement(1544..2494) /codon_start=1 /gene="rep101" /product="RepA protein needed for replication with the pSC101 origin" /label=Rep101 /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI" rep_origin 2542..2764 /label=pSC101 ori /note="low-copy replication origin that requires the Rep101 protein" terminator 3256..3350 /label=lambda t0 terminator /note="transcription terminator from phage lambda" terminator 3506..3535 /label=T3Te terminator /note="phage T3 early transcription terminator" ORIGIN 1 gaattcgttg acagctagct cagtcctagg tataatgcta gctagaagag cgacaggctc 61 ttctgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa 121 aagtggcacc gagtcggtgc tttttttcta gataattgga gaccgagctg gtctctctag 181 gatgtgcact agtagagctc tgcaggagtc actaagggtt agttagttag attagcagaa 241 agtcaaaagc ctccgaccgg aggcttttga ctaaaacttc ccttggggtt atcattgggg 301 ctcactcaaa ggcggtaatc agataaaaaa aatccttagc tttcgctaag gatgatttct 361 gctagtatta ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag 421 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga 481 ggcagttcca aagaatggca aggtcctggt aacggtctgc gattccgacc cgtccaacat 541 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat 601 gagtgacgac tgaatccggt gagaatggca agagcttgtg catttctttc cagacttgtt 661 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca 721 tgcgtgattg cgcctgagca agacgaaata cacgatcgct gttaaaagga caattacaaa 781 caggaatcga atgtaaccgg cgcaggaaca cggccagcgc atcaacaata ttttcacctg 841 aatcaggata ttcttctaat acctggaagg ctgttttccc aggaatcgcg gtggtgagta 901 accacgcatc atcaggagta cggataaaat gcttgatggt cgggagaggc ataaactccg 961 tcagccagtt gagacggacc atctcatctg taacatcatt ggcaacgcta cctttgccat 1021 gtttcagaaa caactctggc gcatcgggct tcccatacaa gcgatagatt gtcgcacctg 1081 attgcccgac attatcgcga gcccatttat acccatataa atcagcgtcc atgttggagt 1141 ttaagcgcgg acgggagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat 1201 tactgtttat gtaagcagac agttttattg ttcatgatga tatattttta tcttgtgcaa 1261 tgtaacatca gagattttga gacacaacgt ggctttgttg aataaatcga acttttgctg 1321 agttgaagga tcagctctag tagttacatt gtcgatctgt tcatggtgaa cagctttgaa 1381 tgcaccaaaa actcgtaaaa gctctgatgt atctatcttt tttacaccgt tttcatctgt 1441 gcatatggac agttttccct ttgatatgta acggtgaaca gttgttctac ttttgtttgt 1501 tagtcttgat gcttcactga tagatacaag agccataaga acctcagatc cttccgtatt 1561 tagccagtat gttctctagt gtggttcgtt gtttttgcgt gagccatgag aacgaaccat 1621 tgagatcata cttactttgc atgtcactca aaaattttgc ctcaaaactg gtgagctgaa 1681 tttttgcagt taaagcatcg tgtagtgttt ttcttagtcc gttatgtagg taggaatctg 1741 atgtaatggt tgttggtatt ttgtcaccat tcatttttat ctggttgttc tcaagttcgg 1801 ttacgagatc catttgtcta tctagttcaa cttggaaaat caacgtatca gtcgggcggc 1861 ctcgcttatc aaccaccaat ttcatattgc tgtaagtgtt taaatcttta cttattggtt 1921 tcaaaaccca ttggttaagc cttttaaact catggtagtt attttcaagc attaacatga 1981 acttaaattc atcaaggcta atctctatat ttgccttgtg agttttcttt tgtgttagtt 2041 cttttaataa ccactcataa atcctcatag agtatttgtt ttcaaaagac ttaacatgtt 2101 ccagattata ttttatgaat ttttttaact ggaaaagata aggcaatatc tcttcactaa 2161 aaactaattc taatttttcg cttgagaact tggcatagtt tgtccactgg aaaatctcaa 2221 agcctttaac caaaggattc ctgatttcca cagttctcgt catcagctct ctggttgctt 2281 tagctaatac accataagca ttttccctac tgatgttcat catctgagcg tattggttat 2341 aagtgaacga taccgtccgt tctttccttg tagggttttc aatcgtgggg ttgagtagtg 2401 ccacacagca taaaattagc ttggtttcat gctccgttaa gtcatagcga ctaatcgcta 2461 gttcatttgc tttgaaaaca actaattcag acatacatct caattggtct aggtgatttt 2521 aatcactata ccaattgaga tgggctagtc aatgataatt acatgtcctt ttcctttgag 2581 ttgtgggtat ctgtaaattc tgctagacct ttgctggaaa acttgtaaat tctgctagac 2641 cctctgtaaa ttccgctaga cctttgtgtg ttttttttgt ttatattcaa gtggttataa 2701 tttatagaat aaagaaagaa taaaaaaaga taaaaagaat agatcccagc cctgtgtata 2761 actcactact ttagtcagtt ccgcagtatt acaaaaggat gtcgcaaacg ctgtttgctc 2821 ctctacaaaa cagaccttaa aaccctaaag gcttaagtag caccctcgca agctcgggca 2881 aatcgctgaa tattcctttt gtctccgacc atcaggcacc tgagtcgctg tctttttcgt 2941 gacattcagt tcgctgcgct cacggctctg gcagtgaatg ggggtaaatg gcactacagg 3001 cgccttttat ggattcatgc aaggaaacta cccataatac aagaaaagcc cgtcacgggc 3061 ttctcagggc gttttatggc gggtctgcta tgtggtgcta tctgactttt tgctgttcag 3121 cagttcctgc cctctgattt tccagtctga ccacttcgga ttatcccgtg acaggtcatt 3181 cagactggct aatgcaccca gtaaggcagc ggtatcatca acaggcttac ccgtcttact 3241 gtccctagtg cttggattct caccaataaa aaacgcccgg cggcaaccga gcgttctgaa 3301 caaatccaga tggagttctg aggtcattac tggatctatc aacaggagtc caagcgagct 3361 cgtaaacttg gtctgacagc tctagctccg gcaaaaaaac gggcaaggtg tcaccaccct 3421 gccctttttc tttaaaaccg aaaagattac ttcgcgtttg ccacctgacg tctaagaaaa 3481 ggaatattca gcaatttgcc cgtgccgaag aaaggcccac ccgtgaaggt gagccagtga 3541 gttgattgct acgtaattag ttagttagcc cttagtgact c //