LOCUS       sequence-308180-        5181 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express sgRNA in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5181)
  AUTHORS   Duan Y, Ma G, Huang X, D'Amore PA, Zhang F, Lei H
  TITLE     The CRISPR/Cas9-created MDM2 T309G enhances vitreous-induced 
            expression of MDM2 and proliferation and survival of cells.
  JOURNAL   J Biol Chem. 2016 May 31. pii: jbc.M116.729467.
  PUBMED    27246850
REFERENCE   2  (bases 1 to 5181)
  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: "J Biol 
            Chem. 2016 May 31. pii: jbc.M116.729467."
COMMENT     Sequence Label: pAAV-U6-sgRNA-CMV-GFP
FEATURES             Location/Qualifiers
     source          1..5181
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     145..165
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     316..335
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        412..487
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        581..884
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        885..1088
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1039..1059
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1127..1136
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1133..1135,1136..1138,1139..1849)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1133..1135,1136..1138,1139..1849)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1178..1199)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1439..1458)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1786..1807
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1928..2404
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2127..2146)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2444..2584
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2444..2573
                     /label=AAV2 ITR
     rep_origin      2659..3114
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2746..2765)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2956..2977
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3131..3150)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3250..3272
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3310..3328)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3396..3500
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3501..3569,3570..4361)
                     /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     complement(3719..3738)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4532..5120
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5021..5040
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accggaagag cgagctcttc tgttttagag
      421 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      481 tcggtgcttt ttttctagag ggtaccgggg cccggtcgac tagttattaa tagtaatcaa
      541 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      601 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      661 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      721 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      781 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      841 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
      901 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
      961 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     1021 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     1081 gcagagctgg tttagtgaac cgtcagatcc gctagcgcta ccggtcgcca ccatggtgag
     1141 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     1201 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     1261 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     1321 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     1381 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     1441 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     1501 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     1561 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     1621 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     1681 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     1741 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     1801 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt ccggactcag
     1861 atctcgataa cctgcagcga attcgatatc aagcttatcg ataccgagcg ctgctcgaga
     1921 gatctacggg tggcatccct gtgacccctc cccagtgcct ctcctggccc tggaagttgc
     1981 cactccagtg cccaccagcc ttgtcctaat aaaattaagt tgcatcattt tgtctgacta
     2041 ggtgtccttc tataatatta tggggtggag gggggtggta tggagcaagg ggcaagttgg
     2101 gaagacaacc tgtagggcct gcggggtcta ttgggaacca agctggagtg cagtggcaca
     2161 atcttggctc actgcaatct ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc
     2221 cgagttgttg ggattccagg catgcatgac caggctcagc taatttttgt ttttttggta
     2281 gagacggggt ttcaccatat tggccaggct ggtctccaac tcctaatctc aggtgatcta
     2341 cccaccttgg cctcccaaat tgctgggatt acaggcgtga accactgctc ccttccctgt
     2401 ccttctgatt ttgtaggtaa ccacgtgcgg accgagcggc cgcaggaacc cctagtgatg
     2461 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     2521 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     2581 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     2641 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2701 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
     2761 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     2821 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     2881 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2941 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     3001 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     3061 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     3121 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     3181 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3241 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     3301 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     3361 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     3421 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     3481 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     3541 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     3601 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     3661 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     3721 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     3781 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     3841 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     3901 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     3961 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     4021 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     4081 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     4141 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     4201 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     4261 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     4321 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     4381 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     4441 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4501 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4561 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4621 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     4681 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     4741 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     4801 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     4861 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4921 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4981 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     5041 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5101 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     5161 gctggccttt tgctcacatg t
//
