pGL3-U6-sgRNA-PGK-puroR-EGFP

LOCUS       pGL3-U6-sgRNA-PGK-puroR-EGFP               5725 bp DNA     circular SYN 13-MAR-2026
DEFINITION  expresses sgRNA from the U6 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5725)
  AUTHORS   Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins 
            A, Iyer V, Huang X, Skarnes WC
  TITLE     Efficient genome modification by CRISPR-Cas9 nickase with minimal 
            off-target effects.
  JOURNAL   Nat Methods. 2014 Mar 2. doi: 10.1038/nmeth.2857.
  PUBMED    24584192
REFERENCE   2  (bases 1 to 5725)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 19, 2026 from SnapGene 6.0.2
            https://www.biofeng.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2014 Mar 2. doi: 10.1038/nmeth.2857."
FEATURES             Location/Qualifiers
     source          1..5725
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        72..312
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     72..92
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     243..262
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        343..418
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    470..587
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        636..1146
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1065..1083
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1168..1764
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1168..1187)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1664..1684
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1765..1821
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     1808..1848
                     /label=EMP39F
     CDS             1822..2541
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     1822..1834
                     /label=N-F
     primer_bind     complement(2513..2541)
                     /label=EMP39R
     primer_bind     complement(2513..2538)
                     /label=N-R
     primer_bind     complement(2517..2538)
                     /label=eGFP_r
     polyA_signal    2688..2809
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2699..2718)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2753..2772
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3057..3074)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3228..3816)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3308..3327)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3987..4847)
                     /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     4610..4629
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4848..4952)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4979..5434
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5066..5085)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5276..5297
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    5565..5613
                     /note="synthetic polyadenylation signal"
     misc_feature    5627..5718
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5667..5686
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg
       61 cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
      121 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
      181 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
      241 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
      301 tgtggaaagg acgaaacacc gtgagaccga gagagggtct cagttttaga gctagaaata
      361 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
      421 tttttaaaga attctcgacc tcgagacaaa tggcagtatt catccacaat tttaaaagaa
      481 aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca
      541 tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca
      601 gggacagcag agatccactt tggccgcggc tcgagggggt tggggttgcg ccttttccaa
      661 ggcagccctg ggtttgcgca gggacgcggc tgctctgggc gtggttccgg gaaacgcagc
      721 ggcgccgacc ctgggactcg cacattcttc acgtccgttc gcagcgtcac ccggatcttc
      781 gccgctaccc ttgtgggccc cccggcgacg cttcctgctc cgcccctaag tcgggaaggt
      841 tccttgcggt tcgcggcgtg ccggacgtga caaacggaag ccgcacgtct cactagtacc
      901 ctcgcagacg gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc
      961 aatagcggct gctcagcagg gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag
     1021 gcggggtgtg gggcggtagt gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc
     1081 aagcctccgg agcgcacgtc ggcagtcggc tccctcgttg accgaatcac cgacctctct
     1141 ccccaggggg atccaccgga gcttaccatg accgagtaca agcccacggt gcgcctcgcc
     1201 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     1261 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     1321 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     1381 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     1441 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     1501 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg
     1561 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     1621 gagcgcgccg gggtgcccgc cttcctggaa acctccgcgc cccgcaacct ccccttctac
     1681 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     1741 tgcatgaccc gcaagcccgg tgccgctact aacttcagcc tgctgaagca ggctggagac
     1801 gtggaggaga accctggacc tatggtgagc aagggcgagg agctgttcac cggggtggtg
     1861 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     1921 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     1981 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
     2041 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     2101 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     2161 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     2221 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     2281 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
     2341 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     2401 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     2461 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     2521 atggacgagc tgtacaagta acgcccgccc cacgacccgc agcgcccgac cgaaaggagc
     2581 gcacgacccc atgcatcggt acctttaaga ccaatgactt acaaggcagc tgtagatctt
     2641 agccactttc tagagtcggg gcggccggcc gcttcgagca gacatgataa gatacattga
     2701 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     2761 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     2821 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta
     2881 aaacctctac aaatgtggta aaatcgataa ggatccgtcg accgatgccc ttgagagcct
     2941 tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga
     3001 ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctcttc cgcttcctcg
     3061 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     3121 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     3181 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3241 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3301 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     3361 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     3421 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     3481 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     3541 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     3601 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     3661 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     3721 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     3781 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     3841 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     3901 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     3961 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4021 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4081 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcggga cccacgctca
     4141 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     4201 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     4261 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     4321 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     4381 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     4441 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     4501 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     4561 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     4621 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     4681 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     4741 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     4801 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     4861 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     4921 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     4981 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     5041 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     5101 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     5161 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     5221 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     5281 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     5341 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     5401 gcgaatttta acaaaatatt aacgcttaca atttgccatt cgccattcag gctgcgcaac
     5461 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagcccaa gctaccatga
     5521 taagtaagta atattaaggt acgggaggta cttggagcgg ccgcaataaa atatctttat
     5581 tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat agtactaaca tacgctctcc
     5641 atcaaaacaa aacgaaacaa aacaaactag caaaataggc tgtccccagt gcaagtgcag
     5701 gtgccagaac atttctctat cgata
//