LOCUS       sequence-77413-e        4952 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses sgRNA from the U6 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4952)
  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 4952)
  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: "Nat 
            Methods. 2014 Mar 2. doi: 10.1038/nmeth.2857."
COMMENT     Sequence Label: pGL3-U6-sgRNA-PGK-puromycin
FEATURES             Location/Qualifiers
     source          1..4952
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        72..320
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     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        344..419
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    471..588
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        637..1147
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1066..1084
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1169..1768
                     /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(1169..1188)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1665..1685
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1915..2036
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1926..1945)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1980..1999
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2284..2301)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2455..3043)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2535..2554)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3214..4005,4006..4074))
                     /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     3837..3856
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4075..4179)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4206..4661
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4293..4312)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4503..4524
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4792..4840
                     /note="synthetic polyadenylation signal"
     misc_feature    4854..4945
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4894..4913
                     /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 ggtgagaccg agagagggtc tcagttttag agctagaaat
      361 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
      421 ttttttaaag aattctcgac ctcgagacaa atggcagtat tcatccacaa ttttaaaaga
      481 aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac
      541 atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac
      601 agggacagca gagatccact ttggccgcgg ctcgaggggg ttggggttgc gccttttcca
      661 aggcagccct gggtttgcgc agggacgcgg ctgctctggg cgtggttccg ggaaacgcag
      721 cggcgccgac cctgggactc gcacattctt cacgtccgtt cgcagcgtca cccggatctt
      781 cgccgctacc cttgtgggcc ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg
      841 ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa gccgcacgtc tcactagtac
      901 cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc
      961 caatagcggc tgctcagcag ggcgcgccga gagcagcggc cgggaagggg cggtgcggga
     1021 ggcggggtgt ggggcggtag tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg
     1081 caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc
     1141 tccccagggg gatccaccgg agcttaccat gaccgagtac aagcccacgg tgcgcctcgc
     1201 cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
     1261 cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga
     1321 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
     1381 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     1441 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     1501 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc
     1561 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     1621 cgagcgcgcc ggggtgcccg ccttcctgga aacctccgcg ccccgcaacc tccccttcta
     1681 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
     1741 gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac gacccgcagc gcccgaccga
     1801 aaggagcgca cgaccccatg catcggtacc tttaagacca atgacttaca aggcagctgt
     1861 agatcttagc cactttctag agtcggggcg gccggccgct tcgagcagac atgataagat
     1921 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     1981 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     2041 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa
     2101 gcaagtaaaa cctctacaaa tgtggtaaaa tcgataagga tccgtcgacc gatgcccttg
     2161 agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca
     2221 cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc gctcttccgc
     2281 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     2341 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     2401 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     2461 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     2521 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     2581 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     2641 gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     2701 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     2761 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     2821 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     2881 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     2941 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     3001 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     3061 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     3121 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     3181 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     3241 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     3301 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgggaccc
     3361 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     3421 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     3481 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     3541 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     3601 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     3661 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     3721 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     3781 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     3841 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     3901 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     3961 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     4021 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     4081 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4141 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     4201 acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     4261 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     4321 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     4381 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     4441 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     4501 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     4561 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     4621 atttaacgcg aattttaaca aaatattaac gtttacaatt tcccattcgc cattcaggct
     4681 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agcccaagct
     4741 accatgataa gtaagtaata ttaaggtacg ggaggtactt ggagcggccg caataaaata
     4801 tctttatttt cattacatct gtgtgttggt tttttgtgtg aatcgatagt actaacatac
     4861 gctctccatc aaaacaaaac gaaacaaaac aaactagcaa aataggctgt ccccagtgca
     4921 agtgcaggtg ccagaacatt tctctatcga ta
//
