LOCUS       sequence-421239-        5948 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Paired gRNAs (sgRNA1 and 2) targeting Exon 1 of VEGFA gene conserved
            across mouse, rhesus macaque, and human..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5948)
  AUTHORS   Chung SH, Sin TN, Dang B, Ngo T, Lo T, Lent-Schochet D, Meleppat RK,
            Zawadzki RJ, Yiu G
  TITLE     CRISPR-based VEGF suppression using paired guide RNAs for treatment 
            of choroidal neovascularization.
  JOURNAL   Mol Ther Nucleic Acids. 2022 Apr 27;28:613-622. doi: 
            10.1016/j.omtn.2022.04.015. eCollection 2022 Jun 14.
  PUBMED    35614998
REFERENCE   2  (bases 1 to 5948)
  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: "Mol Ther 
            Nucleic Acids."; date: "2022-04-27"; volume: "28:613-622. doi"; 
            pages: " 10.1016/j.omtn.2022.04.015. eCollection 2022 Jun 14"
COMMENT     Sequence Label: px552-U6-gRNA1-U6-gRNA2-CMV-eGFP
FEATURES             Location/Qualifiers
     source          1..5948
                     /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)"
     misc_RNA        415..490
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        516..764
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        785..860
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        970..1349
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1351..1549
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1504..1524
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1656..1665
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1662..1664,1665..1667,1668..2378)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1662..1664,1665..1667,1668..2378)
                     /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(1707..1728)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1968..1987)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2315..2336
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2695..3171
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2894..2913)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3211..3351
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3211..3340
                     /label=AAV2 ITR
     rep_origin      3426..3881
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3513..3532)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3723..3744
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3898..3917)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4017..4039
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4077..4095)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4163..4267
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4268..4336,4337..5128)
                     /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(4486..4505)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5299..5887
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5788..5807
                     /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 accgcagaaa gttcatggtt tcgggtttta
      421 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
      481 gagtcggtgc tttttttcta gactgcagag ggcccgaggg cctatttccc atgattcctt
      541 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
      601 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
      661 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
      721 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgagaag tgctagctcg
      781 ggccgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
      841 aagtggcacc gagtcggtgc ttttttctag actgcagagg gcccggcttg accgacaatt
      901 gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat
      961 atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
     1021 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     1081 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     1141 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     1201 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     1261 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     1321 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
     1381 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
     1441 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
     1501 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcagc gcgttttgcc
     1561 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     1621 aacccactgc ttaagccgga tcctctagag tcgacgccac catggtgagc aagggcgagg
     1681 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     1741 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     1801 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     1861 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     1921 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     1981 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     2041 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     2101 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     2161 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     2221 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     2281 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     2341 ccgccgggat cactctcggc atggacgagc tgtacaagtc cggactcaga tctcgagagg
     2401 aggaggagga gacagacagc aggatgcccc acctcgacag ccccggcagc tcccagccga
     2461 gacgctcctt cctctcaagg gtgatcaggg cagcgctacc gttgcagctg cttctgctgc
     2521 tgctgctgct cctggcctgc ctgctacctg cctctgaaga tgactacagc tgcacccagg
     2581 ccaacaactt tgcccgatcc ttctacccca tgctgcggta caccaacggg ccacctccca
     2641 cctaggaatt cgatatcaag cttatcgata ccgagcgctg ctcgagagat ctacgggtgg
     2701 catccctgtg acccctcccc agtgcctctc ctggccctgg aagttgccac tccagtgccc
     2761 accagccttg tcctaataaa attaagttgc atcattttgt ctgactaggt gtccttctat
     2821 aatattatgg ggtggagggg ggtggtatgg agcaaggggc aagttgggaa gacaacctgt
     2881 agggcctgcg gggtctattg ggaaccaagc tggagtgcag tggcacaatc ttggctcact
     2941 gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc agcctcccga gttgttggga
     3001 ttccaggcat gcatgaccag gctcagctaa tttttgtttt tttggtagag acggggtttc
     3061 accatattgg ccaggctggt ctccaactcc taatctcagg tgatctaccc accttggcct
     3121 cccaaattgc tgggattaca ggcgtgaacc actgctccct tccctgtcct tctgattttg
     3181 taggtaacca cgtgcggacc gagcggccgc aggaacccct agtgatggag ttggccactc
     3241 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     3301 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga
     3361 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc
     3421 atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     3481 gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct
     3541 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3601 atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag
     3661 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     3721 tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga
     3781 tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa
     3841 atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac
     3901 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     3961 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     4021 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
     4081 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     4141 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4201 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     4261 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     4321 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     4381 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     4441 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     4501 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     4561 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     4621 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     4681 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     4741 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     4801 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     4861 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     4921 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     4981 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     5041 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     5101 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     5161 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     5221 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     5281 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     5341 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     5401 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     5461 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5521 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5581 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5641 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5701 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5761 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5821 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5881 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5941 tcacatgt
//
