LOCUS       Exported                5958 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AAV vector for U6 driven expression of two gRNAs, and cardiomyocyte 
            specific expression of Cre recombinase..
ACCESSION   .
VERSION     .
KEYWORDS    AAV-U6gRNA1-U6gRNA2-TnT-Cre
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5958)
  AUTHORS   Guo Y, VanDusen NJ, Zhang L, Gu W, Sethi I, Guatimosim S, Ma Q, 
            Jardin BD, Ai Y, Zhang D, Chen B, Guo A, Yuan GC, Song LS, Pu WT
  TITLE     Analysis of Cardiac Myocyte Maturation Using CASAAV, A Platform for 
            Rapid Dissection of Cardiac Myocyte Gene Function In Vivo.
  JOURNAL   Circ Res. 2017 Mar 29. pii: CIRCRESAHA.116.310283. doi: 
            10.1161/CIRCRESAHA.116.310283.
  PUBMED    28356340
REFERENCE   2  (bases 1 to 5958)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5958
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..241
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        278..353
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        366..606
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        633..708
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     intron          1260..1392
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1375..1394
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1437..1455
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1467..1487
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2554..3030
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2753..2772)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3070..3210
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3070..3199
                     /label=AAV2 ITR
     rep_origin      3285..3740
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3372..3391)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3582..3603
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3757..3776)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3876..3898
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3936..3954)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4022..4126
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4127..4987
                     /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(4345..4364)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5158..5746
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5647..5666
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5808..5937
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg actgcaggtg gaattccacc tgctcaggtt ttagagctag aaatagcaag
      301 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgctttttta
      361 cgcgtgaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
      421 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
      481 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
      541 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
      601 aaggacgaaa caccggaaga gcgagctctt ctgttttaga gctagaaata gcaagttaaa
      661 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttctagt
      721 gggataaaag cagtctgggc tttcacatga cagcatctgg ggctgcggca gagggtcggg
      781 tccgaagcgc tgccttatca gcgtccccag ccctgggagg tgacagctgg ctggcttgtg
      841 tcagcccctc gggcactcac gtatctccgt ccgacgggtt taaaatagca aaactctgag
      901 gccacacaat agcttgggct tatatgggct cctgtggggg aagggggagc acggaggggg
      961 ccggggccgc tgctgccaaa atagcagctc acaagtgttg cattcctctc tgggcgccgg
     1021 gcacattcct gctggctctg cccgccccgg ggtgggcgcc ggggggacct taaagcctct
     1081 gccccccaag gagcccttcc cagacagccg ccggcaccca ccgctccgtg ggacgatccc
     1141 cgaagctcta gagctttatt gcggtagttt atcacagtta aattgctaac gcagtcagtg
     1201 cttctgacac aacagtctcg aacttaagct gcagaagttg gtcgtgaggc actgggcagg
     1261 taagtatcaa ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac
     1321 agagaagact cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct
     1381 ttctctccac aggtgtccac tcccagttca attacagctc ttaaggctag agtacttaat
     1441 acgactcact ataggctagc atggtgccca agaagaagag gaaagtctcc aacctgctga
     1501 ctgtgcacca aaacctgcct gccctccctg tggatgccac ctctgatgaa gtcaggaaga
     1561 acctgatgga catgttcagg gacaggcagg ccttctctga acacacctgg aagatgctcc
     1621 tgtctgtgtg cagatcctgg gctgcctggt gcaagctgaa caacaggaaa tggttccctg
     1681 ctgaacctga ggatgtgagg gactacctcc tgtacctgca agccagaggc ctggctgtga
     1741 agaccatcca acagcacctg ggccagctca acatgctgca caggagatct ggcctgcctc
     1801 gcccttctga ctccaatgct gtgtccctgg tgatgaggag aatcagaaag gagaatgtgg
     1861 atgctgggga gagagccaag caggccctgg cctttgaacg cactgacttt gaccaagtca
     1921 gatccctgat ggagaactct gacagatgcc aggacatcag gaacctggcc ttcctgggca
     1981 ttgcctacaa caccctgctg cgcattgccg aaattgccag aatcagagtg aaggacatct
     2041 cccgcaccga tggtgggaga atgctgatcc acattggcag gaccaagacc ctggtgtcca
     2101 cagctggtgt ggagaaggcc ctgtccctgg gggttaccaa gctggtggag agatggatct
     2161 ctgtgtctgg tgtggctgat gaccccaaca actacctgtt ctgccgggtc agaaagaatg
     2221 gtgtggctgc cccttctgcc acctcccaac tgtccacccg ggccctggaa gggatctttg
     2281 aggccaccca ccgcctgatc tatggtgcca aggatgactc tgggcagaga tacctggcct
     2341 ggtctggcca ctctgccaga gtgggtgctg ccagggacat ggccagggct ggtgtgtcca
     2401 tccctgaaat catgcaggct ggtggctgga ccaatgtgaa cattgtgatg aactacatca
     2461 gaaacctgga ctctgagact ggggccatgg tgaggctgct cgaggatggg gactgaaagc
     2521 ttatcgatac cgagcgctgc tcgagagatc tacgggtggc atccctgtga cccctcccca
     2581 gtgcctctcc tggccctgga agttgccact ccagtgccca ccagccttgt cctaataaaa
     2641 ttaagttgca tcattttgtc tgactaggtg tccttctata atattatggg gtggaggggg
     2701 gtggtatgga gcaaggggca agttgggaag acaacctgta gggcctgcgg ggtctattgg
     2761 gaaccaagct ggagtgcagt ggcacaatct tggctcactg caatctccgc ctcctgggtt
     2821 caagcgattc tcctgcctca gcctcccgag ttgttgggat tccaggcatg catgaccagg
     2881 ctcagctaat ttttgttttt ttggtagaga cggggtttca ccatattggc caggctggtc
     2941 tccaactcct aatctcaggt gatctaccca ccttggcctc ccaaattgct gggattacag
     3001 gcgtgaacca ctgctccctt ccctgtcctt ctgattttgt aggtaaccac gtgcggaccg
     3061 agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     3121 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     3181 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc
     3241 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     3301 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3361 cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3421 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3481 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3541 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3601 tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     3661 ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     3721 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     3781 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     3841 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     3901 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     3961 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     4021 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     4081 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     4141 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     4201 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     4261 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     4321 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     4381 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     4441 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     4501 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     4561 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     4621 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     4681 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     4741 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     4801 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     4861 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     4921 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     4981 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     5041 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     5101 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5161 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     5221 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5281 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     5341 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5401 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5461 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5521 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5581 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5641 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5701 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     5761 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgtcct gcaggcagct
     5821 gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc gacctttggt cgcccggcct
     5881 cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg ggttcctgcg
     5941 gccgcacgcg ccggtacc
//
