LOCUS       sequence-434133-        2541 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutively expressed sgRNA targeting a desired gene (this case 
            pta), ColE1 origin, KanR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2541)
  AUTHORS   Mehrer CR, Incha MR, Politz MC, Pfleger BF
  TITLE     Anaerobic production of medium-chain fatty alcohols via a 
            beta-reduction pathway.
  JOURNAL   Metab Eng. 2018 Jul;48:63-71. doi: 10.1016/j.ymben.2018.05.011. Epub
            2018 May 25.
  PUBMED    29807110
REFERENCE   2  (bases 1 to 2541)
  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"; journalName: "Metab 
            Eng."; date: "2018-07"; volume: "48:63-71. doi"; pages: " 
            10.1016/j.ymben.2018.05.011. Epub 2018 May 25"
COMMENT     Sequence Label: pgRNA
FEATURES             Location/Qualifiers
     source          1..2541
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        16..50
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        71..146
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             167..196
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             212..229
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(285..302)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      455..501
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(554..571)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(725..1313)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(805..824)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1484..2299)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2207..2226
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2474..2492
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(2496..2539)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
ORIGIN
        1 gaattctaaa gatctttgac agctagctca gtcctaggta taatactagt caaactggtg
       61 cttacgtgtc gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
      121 ttgaaaaagt ggcaccgagt cggtgctttt tttgaagctt gggcccgaac aaaaactcat
      181 ctcagaagag gatctgaata gcgccgtcga ccatcatcat catcatcatt gagtttaaac
      241 ggtctccagc ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa
      301 tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc
      361 ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg
      421 tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa
      481 agactgggcc tttcgtttta tctgttgttt gtcggtgaac tggatcctta ctcgagtcta
      541 gactgcaggc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
      601 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
      661 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
      721 cgtttttcca caggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
      781 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
      841 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
      901 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
      961 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     1021 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     1081 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     1141 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     1201 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     1261 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     1321 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     1381 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     1441 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagtcagaaa aactcatcga
     1501 gcatcaagtg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     1561 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     1621 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     1681 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     1741 gcaaaagctt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     1801 caaaatcact cgcaccaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     1861 atacgcgatc gccgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     1921 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     1981 atgctgtttt ccctgggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     2041 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagcctg accatctcat
     2101 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     2161 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     2221 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctcgag caagacgttt
     2281 cccgttgaat atggctcatt ttagcttcct tagctcctga aaatctcgat aactcaaaaa
     2341 atacgcccgg tagtgatctt atttcattat ggtgaaagtt ggaacctctt acgtaggggt
     2401 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
     2461 attaacctat aaaaataggc gtatcacgag gcagaatttc agataaaaaa aatccttagc
     2521 tttcgctaag gatgatttct g
//
