LOCUS       sequence-214552-        2665 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA targeting unit plasmid. The sgRNA targeting unit plasmids 
            contain connector ConLS, ConR1, and one sgRNA transcriptional unit. 
            .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2665)
  AUTHORS   Geng P, Leonard SP, Mishler DM, Barrick JE
  TITLE     Synthetic Genome Defenses against Selfish DNA Elements Stabilize 
            Engineered Bacteria against Evolutionary Failure.
  JOURNAL   ACS Synth Biol. 2019 Mar 15;8(3):521-531. doi: 
            10.1021/acssynbio.8b00426. Epub 2019 Feb 15.
  PUBMED    30703321
REFERENCE   2  (bases 1 to 2665)
  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.8b00426"; journalName: "ACS Synth Biol"; date: 
            "2019-03-15- 15"; volume: "8"; issue: "3"; pages: "521-531"
COMMENT     Sequence Label: T5-sgRNA
FEATURES             Location/Qualifiers
     source          1..2665
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     251..269
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(293..307,308..313,314..330,331..337)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    313..329
                     /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)."
     misc_RNA        364..439
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(join(898..1689,1690..1758))
                     /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"
     primer_bind     1521..1540
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1759..1863)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1966..2554
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2455..2474
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaattcgcat ctagatggta gagccacaaa cagccggtac aagcaacgat ctccaggacc
       61 atctgaatca tgcgcggatg acacgaactc acgacggcga tcacagacat taacccacag
      121 tacagacact gcgacaacgt ggcaattcgt cgcaataccg tctcactgaa ctggccgata
      181 attgcagacg aacgcgccct tcccaacagt tgcctaagaa accattatta tcatgacatt
      241 aacctataaa aataggcgta tcacgaggcc ctttcgtctt cacctcgaga aatcataaaa
      301 aatttatttg ctttgtgagc ggataacaat tataatagat tcacatccag ttccaccaga
      361 atagttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
      421 agtggcaccg agtcggtgct ttttttgaat tctctagagt cgacctgcag aagcttagat
      481 ctattaccct gttatcccta ctcgagttca tgtgcagctc catcagcatc cgtaatcgtt
      541 aatccgcaaa taacgtaaaa acccgcttcg gcgggttttt ttatgggggg agtttaggga
      601 aagagcattt gtcagctggc tgccaatgag acgacggggt catcacggct catcatgcgc
      661 caaacaaatg tgtgcaatac acgctcggat gactgcatga tgaccgcact gactggggac
      721 agcagatcca cctaagcctg tgagagaagc agacacccga cagatcaagg cagttaacta
      781 gtgcactgca gtacagcggc cgcgattatc aaaaaggatc ttcacctaga tccttttaaa
      841 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
      901 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
      961 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     1021 tgctgcaatg ataccgcggg acccacgctc accggctcca gatttatcag caataaacca
     1081 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     1141 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     1201 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     1261 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     1321 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     1381 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     1441 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     1501 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     1561 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     1621 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     1681 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     1741 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     1801 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     1861 gcgcacattt ccccgaaaag tgccacctgt catgaccaaa atcccttaac gtgagttttc
     1921 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     1981 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2041 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2101 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2161 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2221 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     2281 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     2341 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     2401 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     2461 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     2521 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     2581 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     2641 tgtggataac cgtgcggccg cccct
//
