LOCUS       sequence-421553-        5578 bp    DNA     circular SYN 15-APR-2025
DEFINITION  sgRNA2 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 5578)
  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 5578)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-gRNA2-CMV-eGFP
FEATURES             Location/Qualifiers
     source          1..5578
                     /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        414..489
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        600..979
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        981..1179
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1134..1154
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1286..1295
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1292..1294,1295..1297,1298..2008)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1292..1294,1295..1297,1298..2008)
                     /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(1337..1358)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1598..1617)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1945..1966
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2325..2801
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2524..2543)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2841..2981
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2841..2970
                     /label=AAV2 ITR
     rep_origin      3056..3511
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3143..3162)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3353..3374
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3528..3547)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3647..3669
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3707..3725)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3793..3897
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3898..3966,3967..4758)
                     /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(4116..4135)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4929..5517
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5418..5437
                     /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 accgagaagt gctagctcgg gccgttttag
      421 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      481 agtcggtgct ttttttctag actgcagagg gcccggcttg accgacaatt gcatgaagaa
      541 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
      601 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
      661 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      721 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      781 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      841 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      901 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      961 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     1021 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     1081 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     1141 gggcggtagg cgtgtacggt gggaggtcta tataagcagc gcgttttgcc tgtactgggt
     1201 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     1261 ttaagccgga tcctctagag tcgacgccac catggtgagc aagggcgagg agctgttcac
     1321 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1381 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     1441 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     1501 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1561 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     1621 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1681 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     1741 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     1801 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     1861 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     1921 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     1981 cactctcggc atggacgagc tgtacaagtc cggactcaga tctcgagagg aggaggagga
     2041 gacagacagc aggatgcccc acctcgacag ccccggcagc tcccagccga gacgctcctt
     2101 cctctcaagg gtgatcaggg cagcgctacc gttgcagctg cttctgctgc tgctgctgct
     2161 cctggcctgc ctgctacctg cctctgaaga tgactacagc tgcacccagg ccaacaactt
     2221 tgcccgatcc ttctacccca tgctgcggta caccaacggg ccacctccca cctaggaatt
     2281 cgatatcaag cttatcgata ccgagcgctg ctcgagagat ctacgggtgg catccctgtg
     2341 acccctcccc agtgcctctc ctggccctgg aagttgccac tccagtgccc accagccttg
     2401 tcctaataaa attaagttgc atcattttgt ctgactaggt gtccttctat aatattatgg
     2461 ggtggagggg ggtggtatgg agcaaggggc aagttgggaa gacaacctgt agggcctgcg
     2521 gggtctattg ggaaccaagc tggagtgcag tggcacaatc ttggctcact gcaatctccg
     2581 cctcctgggt tcaagcgatt ctcctgcctc agcctcccga gttgttggga ttccaggcat
     2641 gcatgaccag gctcagctaa tttttgtttt tttggtagag acggggtttc accatattgg
     2701 ccaggctggt ctccaactcc taatctcagg tgatctaccc accttggcct cccaaattgc
     2761 tgggattaca ggcgtgaacc actgctccct tccctgtcct tctgattttg taggtaacca
     2821 cgtgcggacc gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg
     2881 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     2941 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt
     3001 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     3061 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3121 cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3181 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3241 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     3301 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3361 ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga tttataaggg
     3421 attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3481 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     3541 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     3601 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     3661 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     3721 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     3781 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     3841 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     3901 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     3961 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     4021 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     4081 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     4141 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     4201 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     4261 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     4321 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     4381 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     4441 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     4501 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     4561 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     4621 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     4681 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     4741 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     4801 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     4861 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4921 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4981 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     5041 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     5101 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     5161 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5221 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5281 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5341 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5401 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5461 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     5521 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
