LOCUS       sequence-261181-        6958 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  COL4A3 mutation-specific sgRNA under the control of U6 promoter; 
            mCherry-sgRNA target-(out of frame)EGFP expression cassette; COL4A3 
            donor template.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6958)
  AUTHORS   Daga S, Donati F, Capitani K, Croci S, Tita R, Giliberti A, 
            Valentino F, Benetti E, Fallerini C, Niccheri F, Baldassarri M, 
            Mencarelli MA, Frullanti E, Furini S, Conticello SG, Renieri A, 
            Pinto AM
  TITLE     New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene 
            editing in podocyte-lineage cells.
  JOURNAL   Eur J Hum Genet. 2019 Nov 21. pii: 10.1038/s41431-019-0537-8. doi: 
            10.1038/s41431-019-0537-8.
  PUBMED    31754267
REFERENCE   2  (bases 1 to 6958)
  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: "Eur J Hum 
            Genet. 2019 Nov 21. pii: 10.1038/s41431-019-0537-8. doi: 
            10.1038/s41431-019-0537-8."
COMMENT     Sequence Label: pAAV_Donor/Reporter-COL4A3
FEATURES             Location/Qualifiers
     source          1..6958
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   259..388
                     /label=AAV2 ITR
     enhancer        654..957
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        958..1161
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1112..1132
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1198..1905
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1343..1361)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      2001..2010
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2007..2009,2010..2012,2013..2726)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2007..2009,2010..2012,2013..2723)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2052..2073)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2313..2332)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2660..2681
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2799..2819)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3217..3228)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3295..3312)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3301..3508
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    3639..3720
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3676..3695)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3737..3985
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3737..3757
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3908..3927
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4007..4082
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     repeat_region   4266..4395
                     /label=AAV2 ITR
     primer_bind     complement(4414..4431)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4414..4430)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4423..4445)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4572..5027
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4659..4678)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4869..4890
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5053..5157
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5158..5226,5227..6018)
                     /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(5376..5395)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6189..6777
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6678..6697
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta
      241 attaaggcct taattaggct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc
      301 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga
      361 gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta
      421 tctacgtagc catgctctag gaagatcgga attcgccctt aaagacccag gaattccagg
      481 cttggataga tcaggatttc ctggagaaac tggatcacca ggaattccag gtcatcaagg
      541 tgaaatggga ccactgggtc aatcgacggt atcgaattcg cccttaagct agctagttat
      601 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
      661 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      721 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      781 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      841 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
      901 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
      961 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
     1021 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
     1081 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
     1141 gaggtctata taagcagagc tggtttagtg aaccgtcaga tccctagcgg attcaccatg
     1201 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
     1261 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     1321 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     1381 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     1441 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     1501 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     1561 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     1621 cagaaaaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
     1681 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
     1741 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
     1801 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
     1861 gccgagggcc gccactccac cggcggcatg gacgagctgt acaagaagct tcgtctctgt
     1921 cgcctggaga aactgaatca cctggtctgc gagacggaat tctgcaatcg acggtaccac
     1981 gggcccggga tccaccggtc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg
     2041 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     2101 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     2161 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     2221 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2281 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     2341 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     2401 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     2461 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     2521 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     2581 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     2641 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     2701 tcggcatgga cgagctgtac aagtaaagcg gccgcgcttg gatccaatca acctctggat
     2761 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     2821 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     2881 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     2941 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     3001 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     3061 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     3121 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
     3181 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     3241 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgaga tctgcctcga
     3301 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     3361 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     3421 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     3481 gggaagacaa tagcaggcat gctggggact cgagttagcg gccgcgactc tagatcataa
     3541 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
     3601 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
     3661 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     3721 attctagttg ccgcgggagg gcctatttcc catgattcct tcatatttgc atatacgata
     3781 caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca
     3841 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     3901 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     3961 tatcttgtgg aaaggacgaa acaccgcctg gagaaactga atcaccgttt tagagctaga
     4021 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     4081 gcttttttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc
     4141 gtgcgccaat tctgcagaca aatggctcta gaggtaccct cgagttaagg gcgaattccc
     4201 gataaggatc ttcctagagc atggctacgt agataagtag catggcgggt taatcattaa
     4261 ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     4321 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     4381 cgagcgagcg cgcagcctta attaacctaa ttcactggcc gtcgttttac aacgtcgtga
     4441 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     4501 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     4561 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4621 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     4681 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     4741 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     4801 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4861 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     4921 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     4981 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     5041 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5101 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5161 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5221 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5281 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5341 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5401 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5461 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5521 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5581 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5641 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5701 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5761 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5821 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     5881 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     5941 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6001 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6061 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6121 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6181 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6241 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6301 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6361 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6421 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6481 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6541 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6601 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6661 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6721 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6781 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     6841 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     6901 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaag
//
