LOCUS       Exported                5460 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  An AAV genome with tet-inducible, Cre-dependent expression of the 
            fluorescent protein eYFP.
ACCESSION   .
VERSION     .
KEYWORDS    TRE-DIO-eYFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5460)
  AUTHORS   Challis RC, Ravindra Kumar S, Chan KY, Challis C, Beadle K, Jang MJ,
            Kim HM, Rajendran PS, Tompkins JD, Shivkumar K, Deverman BE, 
            Gradinaru V
  TITLE     Systemic AAV vectors for widespread and targeted gene delivery in 
            rodents.
  JOURNAL   Nat Protoc. 2019 Feb;14(2):379-414. doi: 10.1038/s41596-018-0097-3.
  PUBMED    30626963
REFERENCE   2  (bases 1 to 5460)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5460
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    165..183
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    202..220
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    239..257
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    276..294
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    313..331
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    350..368
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    387..405
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    424..442
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     464..488
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    587..620
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    671..704
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(713..1432)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(758..779)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1107..1126
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1366..1387
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1429..1438
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(1445..1478)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    complement(1529..1562)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    1587..2175
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1640..1660)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2058..2069)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2207..2683
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2406..2425)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2723..2863
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2723..2852
                     /label=AAV2 ITR
     rep_origin      2938..3393
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3025..3044)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3235..3256
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3410..3429)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3529..3551
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3589..3607)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3675..3779
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3780..4640
                     /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(3998..4017)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4811..5399
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5300..5319
                     /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 gcgtatcaag tgccacctga cgtctcccta tcagtgatag
      181 agaagtcgac acgtctcgag ctccctatca gtgatagaga aggtacgtct agaacgtctc
      241 cctatcagtg atagagaagt cgacacgtct cgagctccct atcagtgata gagaaggtac
      301 gtctagaacg tctccctatc agtgatagag aagtcgacac gtctcgagct ccctatcagt
      361 gatagagaag gtacgtctag aacgtctccc tatcagtgat agagaagtcg acacgtctcg
      421 agctccctat cagtgataga gaagctaccc cctatataag cagagctcgt ttagtgaacc
      481 gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc
      541 gatccagcct ggatccggta ccggatcctc tagagtcgac tccggaataa cttcgtatag
      601 gatactttat acgaagttat gcagaatggt agctggattg tagctgctat tagcaatatg
      661 aaacctctta ataacttcgt atagcataca ttatacgaag ttatggcgcg ccttacttgt
      721 acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt
      781 gatcgcgctt ctcgttgggg tctttgctca gggcggactg gtagctcagg tagtggttgt
      841 cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct
      901 gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg ccgttcttct
      961 gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc ttgtgcccca
     1021 ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt
     1081 cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg
     1141 tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt
     1201 ggtcggggta gcgggcgaag cactgcaggc cgtagccgaa ggtggtcacg agggtgggcc
     1261 agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg
     1321 catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct
     1381 cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc atggtggcgc
     1441 tagcataact tcgtataaag tatcctatac gaagttattt gccttaaccc agaaattatc
     1501 actgttattc tttagaatgg tgcaaagaat aacttcgtat aatgtatgct atacgaagtt
     1561 atgaattcga tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat
     1621 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     1681 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     1741 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     1801 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     1861 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     1921 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     1981 aatcatcgtc ctttccttgg ctgctcgcct atgttgccac ctggattctg cgcgggacgt
     2041 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     2101 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     2161 gggccgcctc cccgcatcga taccgagcgc tgctcgagag atctacgggt ggcatccctg
     2221 tgacccctcc ccagtgcctc tcctggccct ggaagttgcc actccagtgc ccaccagcct
     2281 tgtcctaata aaattaagtt gcatcatttt gtctgactag gtgtccttct ataatattat
     2341 ggggtggagg ggggtggtat ggagcaaggg gcaagttggg aagacaacct gtagggcctg
     2401 cggggtctat tgggaaccaa gctggagtgc agtggcacaa tcttggctca ctgcaatctc
     2461 cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc gagttgttgg gattccaggc
     2521 atgcatgacc aggctcagct aatttttgtt tttttggtag agacggggtt tcaccatatt
     2581 ggccaggctg gtctccaact cctaatctca ggtgatctac ccaccttggc ctcccaaatt
     2641 gctgggatta caggcgtgaa ccactgctcc cttccctgtc cttctgattt tgtaggtaac
     2701 cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg
     2761 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     2821 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat
     2881 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
     2941 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     3001 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     3061 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     3121 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
     3181 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     3241 tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt gatttataag
     3301 ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     3361 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct
     3421 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     3481 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     3541 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     3601 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     3661 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     3721 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     3781 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     3841 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     3901 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     3961 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     4021 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     4081 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     4141 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     4201 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     4261 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     4321 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     4381 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     4441 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     4501 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     4561 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     4621 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     4681 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     4741 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     4801 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     4861 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     4921 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     4981 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     5041 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     5101 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     5161 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     5221 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     5281 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     5341 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     5401 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
