LOCUS       Exported                6953 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  COL4A5 mutation-specific sgRNA under the control of U6 promoter; 
            mCherry-sgRNA target-(out of frame)EGFP expression cassette; COL4A5 
            donor template.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV_Donor/Reporter-COL4A5
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6953)
  TITLE     New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene 
            editing in podocyte-lineage cells
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6953)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6953
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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        143..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        649..952
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        953..1156
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1107..1127
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1193..1900
                     /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(1338..1356)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      1996..2005
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2002..2721
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQSTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2047..2068)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2308..2327)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2655..2676
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2794..2814)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3212..3223)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3290..3307)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3296..3503
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    3634..3715
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3671..3690)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3732..3972
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     3732..3752
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3903..3922
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4002..4077
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     repeat_region   4261..4390
                     /label=AAV2 ITR
     primer_bind     complement(4409..4426)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4409..4425)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4418..4440)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4567..5022
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4654..4673)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4864..4885
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5048..5152
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5153..6013
                     /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(5371..5390)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6184..6772
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6673..6692
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6926..6943
                     /label=L4440
                     /note="L4440 vector, 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 aagcccaggt ttaccaggcc
      481 tcccagggaa tatagggcct atgggtcccc ctggtttcgg ccctccaggc ccagtaggtg
      541 aaaaaggcat acaaggtgtg gcaggtcgaa attcgccctt aagctagcta gttattaata
      601 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
      661 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
      721 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
      781 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
      841 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
      901 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
      961 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     1021 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     1081 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
     1141 ctatataagc agagctggtt tagtgaaccg tcagatccct agcggattca ccatggtgag
     1201 caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat
     1261 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     1321 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg
     1381 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
     1441 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     1501 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     1561 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     1621 aaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
     1681 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa
     1741 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa
     1801 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga
     1861 gggccgccac tccaccggcg gcatggacga gctgtacaag aagcttcgtc tctgtcgcct
     1921 ggagggccga aatcaggggg tctgcgagac ggaattctgc aatcgacggt accacgggcc
     1981 cgggatccac cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg
     2041 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     2101 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     2161 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
     2221 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     2281 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     2341 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     2401 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     2461 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
     2521 gacggcagcg tgcagctcgc cgaccactac cagcagagca cccccatcgg cgacggcccc
     2581 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     2641 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     2701 atggacgagc tgtacaagta aagcggccgc gcttggatcc aatcaacctc tggattacaa
     2761 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     2821 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     2881 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     2941 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     3001 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     3061 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     3121 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat
     3181 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     3241 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgagatctgc ctcgactgtg
     3301 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     3361 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     3421 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     3481 gacaatagca ggcatgctgg ggactcgagt tagcggccgc gactctagat cataatcagc
     3541 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
     3601 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
     3661 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     3721 agttgccgcg ggagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     3781 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
     3841 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     3901 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     3961 tgtggaaagg acgaaacacc gcctggaggg ccgaaatcag ggttttagag ctagaaatag
     4021 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     4081 tttgttttag agctagaaat agcaagttaa aataaggcta gtccgttttt agcgcgtgcg
     4141 ccaattctgc agacaaatgg ctctagaggt accctcgagt taagggcgaa ttcccgataa
     4201 ggatcttcct agagcatggc tacgtagata agtagcatgg cgggttaatc attaactaca
     4261 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     4321 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     4381 gagcgcgcag ccttaattaa cctaattcac tggccgtcgt tttacaacgt cgtgactggg
     4441 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     4501 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     4561 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     4621 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     4681 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     4741 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     4801 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4861 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     4921 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     4981 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     5041 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5101 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5161 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5221 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     5281 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     5341 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     5401 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     5461 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     5521 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     5581 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     5641 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     5701 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     5761 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     5821 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     5881 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     5941 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6001 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     6061 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6121 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     6181 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     6241 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     6301 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     6361 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     6421 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     6481 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     6541 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6601 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     6661 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     6721 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     6781 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     6841 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     6901 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aag
//
