LOCUS       sequence-140493-        3581 bp    DNA     circular SYN 15-DEC-2025
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    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acssynbio.5b00132"; journalName: "ACS Synth Biol"; date: 
            "2015-11-20- 20"; volume: "4"; issue: "11"; pages: "1217-25"
COMMENT     Sequence Label: pSG4K5
FEATURES             Location/Qualifiers
     source          1..3581
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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      complement(3256..3350)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      complement(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
//
