LOCUS       sequence-273618-        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"
     promoter        64..312
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        331..406
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(505..521)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(505..521)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(518..540)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    529..545
                     /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(553..559,560..577,578..583))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    598..619
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(736..753)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(907..1495)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(987..1006)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1666..2457,2458..2526))
                     /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     2289..2308
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2527..2631)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2699..2717
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2755..2777)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2877..2896
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3090..3112
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3104..3121
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3105..3121
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3128..3376
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        3398..3473
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
       61 agagagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
      121 gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
      181 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
      241 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
      301 ggacgaaaca ccgggtcttc gagaagacct gttttagagc tagaaatagc aagttaaaat
      361 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga
      421 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca
      481 gacaaaaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
      541 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
      601 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
      661 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
      721 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
      781 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
      841 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
      901 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
      961 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     1021 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     1081 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     1141 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     1201 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     1261 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     1321 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     1381 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     1441 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     1501 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     1561 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     1621 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     1681 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     1741 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     1801 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     1861 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     1921 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     1981 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     2041 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     2101 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     2161 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     2221 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     2281 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     2341 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     2401 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     2461 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     2521 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     2581 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     2641 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     2701 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
     2761 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     2821 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
     2881 atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt
     2941 aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg
     3001 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
     3061 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
     3121 tgaattcgag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
     3181 tagagagata attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
     3241 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
     3301 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
     3361 gaaaggacga aacaccgacc cacggcgtgc agtgcttgtt ttagagctag aaatagcaag
     3421 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg
     3481 ttttagagct
//
