LOCUS       sequence-421556-        5579 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA1 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 5579)
  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 5579)
  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-CMV-eGFP
FEATURES             Location/Qualifiers
     source          1..5579
                     /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        415..490
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        601..980
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        982..1180
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1135..1155
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1287..1296
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1293..1295,1296..1298,1299..2009)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1293..1295,1296..1298,1299..2009)
                     /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(1338..1359)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1599..1618)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1946..1967
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2326..2802
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2525..2544)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2842..2982
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2842..2971
                     /label=AAV2 ITR
     rep_origin      3057..3512
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3144..3163)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3354..3375
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3529..3548)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3648..3670
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3708..3726)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3794..3898
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3899..3967,3968..4759)
                     /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(4117..4136)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4930..5518
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5419..5438
                     /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 ggcccggctt gaccgacaat tgcatgaaga
      541 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
      601 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
      661 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      721 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      781 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      841 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      901 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      961 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
     1021 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     1081 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
     1141 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
     1201 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     1261 cttaagccgg atcctctaga gtcgacgcca ccatggtgag caagggcgag gagctgttca
     1321 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     1381 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     1441 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     1501 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     1561 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     1621 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     1681 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     1741 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     1801 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     1861 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
     1921 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     1981 tcactctcgg catggacgag ctgtacaagt ccggactcag atctcgagag gaggaggagg
     2041 agacagacag caggatgccc cacctcgaca gccccggcag ctcccagccg agacgctcct
     2101 tcctctcaag ggtgatcagg gcagcgctac cgttgcagct gcttctgctg ctgctgctgc
     2161 tcctggcctg cctgctacct gcctctgaag atgactacag ctgcacccag gccaacaact
     2221 ttgcccgatc cttctacccc atgctgcggt acaccaacgg gccacctccc acctaggaat
     2281 tcgatatcaa gcttatcgat accgagcgct gctcgagaga tctacgggtg gcatccctgt
     2341 gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt
     2401 gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta taatattatg
     2461 gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc
     2521 ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc
     2581 gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca
     2641 tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg
     2701 gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg
     2761 ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt gtaggtaacc
     2821 acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     2881 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     2941 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     3001 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     3061 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3121 acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3181 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3241 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     3301 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3361 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg
     3421 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3481 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     3541 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     3601 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     3661 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     3721 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     3781 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     3841 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     3901 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     3961 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     4021 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     4081 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     4141 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     4201 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     4261 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     4321 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     4381 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     4441 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     4501 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     4561 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     4621 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     4681 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     4741 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     4801 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4861 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4921 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4981 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5041 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     5101 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5161 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     5221 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     5281 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5341 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5401 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     5461 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     5521 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
