LOCUS       Exported                6292 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AAV backbone with a full length U6/TO promoter driving expression of
            a sgRNA that targets exon 3 of the mouse Tet2 locus. TracrRNA 
            compatible with SpCas9. gRNA transcription is doxycycline 
            dependent..
ACCESSION   .
VERSION     .
KEYWORDS    pJEP11-AAV-U6/TO-sgRNA(Tet2)-CMV-TetR-P2A-eGFP-KASH-pA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6292)
  AUTHORS   de Solis CA, Ho A, Holehonnur R, Ploski JE
  TITLE     The Development of a Viral Mediated CRISPR/Cas9 System with 
            Doxycycline Dependent gRNA Expression for Inducible In vitro and In 
            vivo Genome Editing.
  JOURNAL   Front Mol Neurosci. 2016 Aug 18;9:70. doi: 10.3389/fnmol.2016.00070.
            eCollection 2016.
  PUBMED    27587996
REFERENCE   2  (bases 1 to 6292)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6292
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(113..132)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     460..481
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    815..1403
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(868..888)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1286..1297)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   1490..1630
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1490..1619
                     /label=AAV2 ITR
     rep_origin      1705..2160
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1792..1811)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2002..2023
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2177..2196)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2296..2318
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2356..2374)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2442..2546
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2547..3407
                     /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(2765..2784)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3578..4166
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4067..4086
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   4228..4357
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     4372..4392
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4543..4562
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    4573..4591
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4601..4619
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        4642..4717
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        4751..5054
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5055..5258
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5209..5229
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5255..5279
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             5409..6026
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG"
     primer_bind     complement(5482..5501)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             6048..6101
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             6102..523
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(6144..6165)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
ORIGIN
        1 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
       61 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
      121 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
      181 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
      241 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
      301 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
      361 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
      421 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
      481 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtccggac tcagatctcg
      541 agaggaggag gaggagacag acagcaggat gccccacctc gacagccccg gcagctccca
      601 gccgagacgc tccttcctct caagggtgat cagggcagcg ctaccgttgc agctgcttct
      661 gctgctgctg ctgctcctgg cctgcctgct acctgcctct gaagatgact acagctgcac
      721 ccaggccaac aactttgccc gatccttcta ccccatgctg cggtacacca acgggccacc
      781 tcccacctag gaattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg
      841 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
      901 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
      961 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     1021 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     1081 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     1141 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     1201 gtcggggaaa tcatcgtcct ttccttggct gctcgcctat gttgccacct ggattctgcg
     1261 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     1321 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     1381 ctccctttgg gccgcctccc cgcatcgata ccgagcgcta ataaaagatc tttattttca
     1441 ttagatctgt gtgttggttt tttgtgtcac gtgcggaccg agcggccgca ggaaccccta
     1501 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     1561 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     1621 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     1681 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1741 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg
     1801 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     1861 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     1921 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     1981 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta
     2041 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa
     2101 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     2161 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     2221 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     2281 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     2341 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     2401 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     2461 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     2521 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     2581 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     2641 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     2701 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     2761 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     2821 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     2881 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     2941 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     3001 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     3061 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     3121 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     3181 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     3241 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     3301 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     3361 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     3421 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     3481 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     3541 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3601 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3661 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3721 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3781 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3841 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3901 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3961 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4021 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4081 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4141 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4201 ccttttgctg gccttttgct cacatgtcct gcaggcagct gcgcgctcgc tcgctcactg
     4261 aggccgcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga gcgagcgcgc
     4321 agagagggag tggccaactc catcactagg ggttcctgcg gccgcacgcg tgagggccta
     4381 tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga
     4441 attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa
     4501 tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc
     4561 gtaacttgaa actccctatc agtgatagag attatatatc tccctatcag tgatagagac
     4621 cgaaagtgcc aacagatatc cgttttagag ctagaaatag caagttaaaa taaggctagt
     4681 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttaccggtc catggactag
     4741 ttctagagtc cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     4801 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     4861 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     4921 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     4981 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     5041 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
     5101 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
     5161 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
     5221 gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg
     5281 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
     5341 tccgcggccg ggaacggtgc attggaacgg acctgcagga attgatccgc ggccgcaccg
     5401 gtaccatgat gtccagatta gataaaagta aagtgattaa cagcgcatta gagctgctta
     5461 atgaggtcgg aatcgaaggt ttaacaaccc gtaaactcgc ccagaagcta ggtgtagagc
     5521 agcctacatt gtattggcat gtaaaaaata agcgggcttt gctcgacgcc ttagccattg
     5581 agatgttaga taggcaccat actcactttt gccctttaga aggggaaagc tggcaagatt
     5641 ttttacgtaa taacgctaaa agttttagat gtgctttact aagtcatcgc gatggagcaa
     5701 aagtacattt aggtacacgg cctacagaaa aacagtatga aactctcgaa aatcaattag
     5761 cctttttatg ccaacaaggt ttttcactag agaatgcatt atatgcactc agcgctgtgg
     5821 ggcattttac tttaggttgc gtattggaag atcaagagca tcaagtcgct aaagaagaaa
     5881 gggaaacacc tactactgat agtatgccgc cattattacg acaagctatc gaattatttg
     5941 atcaccaagg tgcagagcca gccttcttat tcggccttga attgatcata tgcggattag
     6001 aaaaacaact taaatgtgaa agtgggtccg cggctagcgg cagtggagag ggcagaggaa
     6061 gtctgctaac atgcggtgac gtcgaggaga atcctggccc agtgagcaag ggcgaggagc
     6121 tgttcaccgg ggtggtaccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     6181 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     6241 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ct
//
