LOCUS       sequence-218814-        2661 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 2661)
  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 2661)
  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..2661
                     /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(894..1685,1686..1754))
                     /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     1517..1536
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1755..1859)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1962..2550
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2451..2470
                     /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 gtcagctgcc aatgagacga cggggtcatc acggctcatc atgcgccaaa
      661 caaatgtgtg caatacacgc tcggatgact gcatgatgac cgcactgact ggggacagca
      721 gatccaccta agcctgtgag agaagcagac acccgacaga tcaaggcagt taactagtgc
      781 actgcagtac agcggccgcg attatcaaaa aggatcttca cctagatcct tttaaattaa
      841 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
      901 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
      961 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     1021 gcaatgatac cgcgggaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     1081 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     1141 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     1201 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     1261 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     1321 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     1381 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     1441 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     1501 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     1561 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     1621 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     1681 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     1741 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     1801 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     1861 acatttcccc gaaaagtgcc acctgtcatg accaaaatcc cttaacgtga gttttcgttc
     1921 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     1981 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     2041 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     2101 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     2161 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     2221 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcggactga
     2281 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     2341 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     2401 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     2461 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     2521 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     2581 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     2641 gataaccgtg cggccgcccc t
//
