LOCUS       sequence-266850-        3490 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual-targeting sgRNA vector that contains both a sgRNA against BFP 
            (sgBG) and a sgRNA for a genomic target site (sgTS).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3490)
  AUTHORS   Standage-Beier K, Tekel SJ, Brookhouser N, Schwarz G, Nguyen T, Wang
            X, Brafman DA
  TITLE     A transient reporter for editing enrichment (TREE) in human cells.
  JOURNAL   Nucleic Acids Res. 2019 Aug 20. pii: 5552066. doi: 
            10.1093/nar/gkz713.
  PUBMED    31428784
REFERENCE   2  (bases 1 to 3490)
  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: "Nucleic 
            Acids Res. 2019 Aug 20. pii: 5552066. doi: 10.1093/nar/gkz713."
COMMENT     Sequence Label: pDT-sgRNA
FEATURES             Location/Qualifiers
     source          1..3490
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        402..650
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        672..747
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        828..1076
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1095..1170
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1269..1285)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1269..1285)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1282..1304)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1293..1309
                     /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)."
     promoter        complement(join(1317..1323,1324..1341,1342..1347))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1362..1383
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1500..1517)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1671..2259)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1751..1770)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2430..3221,3222..3290))
                     /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     3053..3072
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3291..3395)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3463..3481
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagggccta tttcccatga
      421 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga
      481 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
      541 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
      601 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gacccacggc
      661 gtgcagtgct tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
      721 cttgaaaaag tggcaccgag tcggtgcttt tttgttttag agctagaaat agcaagttaa
      781 aataaggcta gtccgttttt agcgcgtgcg ccaattctgc agacagagag ggcctatttc
      841 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attggaatta
      901 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
      961 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     1021 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgggt
     1081 cttcgagaag acctgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
     1141 caacttgaaa aagtggcacc gagtcggtgc ttttttgttt tagagctaga aatagcaagt
     1201 taaaataagg ctagtccgtt tttagcgcgt gcgccaattc tgcagacaaa aagcttggcg
     1261 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     1321 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     1381 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     1441 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     1501 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     1561 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     1621 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     1681 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     1741 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     1801 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     1861 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     1921 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     1981 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     2041 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     2101 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     2161 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     2221 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     2281 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     2341 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     2401 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     2461 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     2521 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     2581 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     2641 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     2701 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     2761 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     2821 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     2881 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     2941 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     3001 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     3061 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     3121 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     3181 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     3241 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     3301 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     3361 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     3421 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     3481 ccctttcgtc
//
