LOCUS       662_pAAV-Ef1a-fDIO-rc[C 7335 bp ds-DNA     circular     15-MAR-2020
DEFINITION  .
COMMENT     PCR-cloning of EGFP from AAV-Cre-GFP (Addgene #68544) into
            pAAV-Ef1a-fDIO EYFP (Addgene #55641) by BsrGI-NcoI
FEATURES             Location/Qualifiers
     promoter        249..1419
                     /label="EF1a_promoter"
     primer          1247..1267
                     /label="224565890_EZ_Ef1a_Fwd"
                     /note="sequence: ggttttatgcgatggagtttc"
     misc_feature    1367..1387
                     /label="EF1a_fwd_primer"
     primer          1420..1440
                     /label="222188113_OR_AAV-fDIO_Gibson-7"
                     /note="sequence: ggtaccggatcctctagagtc"
     misc_feature    1450..1483
                     /label="FRT"
     misc_feature    1534..1567
                     /label="FRT2"
     primer          complement(1552..1571)
                     /label="OR_AAV-fDIO_Gibson-1"
                     /note="sequence: cgccgaagttcctatacctt"
     misc_feature    1574..1586
                     /label="MT_AAV-fDIO-EGFP-1"
     CDS             complement(1576..1580)
                     /label="ORF frame 3"
     CDS             1576..2292
                     /label="EGFP"
     misc_feature    complement(1576..3285)
                     /label="Chronos-GFP"
     CDS             complement(1576..3285)
                     /label="Translation 3285-1576"
                     /translation="METAATMTHAFISAVPSAEATIRGLLSAAAVVTPAADAHGETSNATTAGADHGCFPHINHGTELQHKIAVGLQWFTVIVAIVQLIFYGWHSFKATTGWEEVYVCVIELVKCFIELFHEVDSPATVYQTNGGAVIWLRYSMWLLTCPVILIHLSNLTGLHEEYSKRTMTILVTDIGNIVWGITAAFTKGPLKILFFMIGLFYGVTCFFQIAKVYIESYHTLPKGVCRKICKIMAYVFFCSWLMFPVMFIAGHEGLGLITPYTSGIGHLILDLISKNTWGFLGHHLRVKIHEHILIHGDIRKTTTINVAGENMEIETFVDEEEEGGVAAPVVAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*"
     misc_feature    1584..3284
                     /label="Insert (origin: p#645)"
     primer_bind     complement(1621..1642)
                     /label="EGFP-C"
     primer          1772..1790
                     /label="224565891_EZ_Chronos-eGFP_Fwd1"
                     /note="sequence: CCGTCCTCGATGTTGTGGC"
     primer_bind     1970..1989
                     /label="EXFP-R"
     primer          complement(2077..2099)
                     /label="71992159_IS_GenSat_EGFP_For"
                     /note="sequence: CCTACGGCGTGCAGTGCTTCAGC"
     primer          2144..2164
                     /label="224565892_EZ_Chronos-eGFP_Fwd2"
                     /note="sequence: GTGGTGCAAATGAACTTCAGG"
     primer_bind     2229..2250
                     /label="EGFP-N"
     primer          2515..2532
                     /label="224565893_EZ_Chronos-eGFP_Fwd3"
                     /note="sequence: GATCAGGCCCAGTCCCTC"
     primer          2864..2882
                     /label="224565894_EZ_Chronos-eGFP_Fwd4"
                     /note="sequence: CACATGCTGTACCGCAGCC"
     primer          3183..3201
                     /label="224565895_EZ_Chronos-eGFP_Fwd5"
                     /note="sequence: CTGGTGTCACCACTGCTGC"
     misc_feature    complement(3282..3285)
                     /label="Priming-site"
     misc_feature    complement(3282..3291)
                     /label="MT_AAV-fDIO-EGFP-2"
     misc_feature    3285..1583
                     /label="Vector (origin: p#574)"
     primer          3290..3309
                     /label="OR_AAV-fDIO_Gibson-2"
                     /note="sequence: gcgctagcgaagttcctata"
     misc_feature    complement(3298..3331)
                     /label="FRT"
     misc_feature    complement(3382..3415)
                     /label="FRT2"
     misc_feature    3442..4051
                     /label="WPRE"
     primer          complement(3897..3914)
                     /label="77631386_OR_WPRE_R_Tit"
                     /note="sequence: ccgaagggacgtagcaga"
     terminator      4080..4558
                     /label="hGH_PA_terminator"
     primer          complement(4539..4556)
                     /label="224592105_EZ_hGH-PA-Terminator_Rev"
                     /note="sequence: ggacagggaagggagcag"
     rep_origin      4830..5136
                     /label="f1_origin"
     misc_feature    5404..5426
                     /label="pGEX_3_primer"
     promoter        5585..5613
                     /label="AmpR_promoter"
     CDS             5655..6515
                     /label="ORF frame 2"
     gene            5655..6515
                     /label="Ampicillin"
     rep_origin      6670..7289
                     /label="pBR322_origin"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt
      181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
      241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
      481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
      541 tacttccact ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      601 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      661 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      721 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      781 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      841 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      901 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      961 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     1021 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     1081 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     1141 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     1201 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1261 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1321 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1381 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc
     1441 gactccggag aagttcctat tctctagaaa gtataggaac ttcgcagaat ggtagctgga
     1501 ttgtagctgc tattagcaat atgaaacctc ttagaagttc ctattcttca aaaggtatag
     1561 gaacttcggc gcgccTTACT TGTACAGCTC GTCCATGCCG AGAGTGATCC CGGCGGCGGT
     1621 CACGAACTCC AGCAGGACCA TGTGATCGCG CTTCTCGTTG GGGTCTTTGC TCAGGGCGGA
     1681 CTGGGTGCTC AGGTAGTGGT TGTCGGGCAG CAGCACGGGG CCGTCGCCGA TGGGGGTGTT
     1741 CTGCTGGTAG TGGTCGGCGA GCTGCACGCT GCCGTCCTCG ATGTTGTGGC GGATCTTGAA
     1801 GTTCACCTTG ATGCCGTTCT TCTGCTTGTC GGCCATGATA TAGACGTTGT GGCTGTTGTA
     1861 GTTGTACTCC AGCTTGTGCC CCAGGATGTT GCCGTCCTCC TTGAAGTCGA TGCCCTTCAG
     1921 CTCGATGCGG TTCACCAGGG TGTCGCCCTC GAACTTCACC TCGGCGCGGG TCTTGTAGTT
     1981 GCCGTCGTCC TTGAAGAAGA TGGTGCGCTC CTGGACGTAG CCTTCGGGCA TGGCGGACTT
     2041 GAAGAAGTCG TGCTGCTTCA TGTGGTCGGG GTAGCGGCTG AAGCACTGCA CGCCGTAGGT
     2101 CAGGGTGGTC ACGAGGGTGG GCCAGGGCAC GGGCAGCTTG CCGGTGGTGC AAATGAACTT
     2161 CAGGGTCAGC TTGCCGTAGG TGGCATCGCC CTCGCCCTCG CCGGACACGC TGAACTTGTG
     2221 GCCGTTTACG TCGCCGTCCA GCTCGACCAG GATGGGCACC ACCCCGGTGA ACAGCTCCTC
     2281 GCCCTTGCTC ACTGCTACTA CCGGTGCCGC CACTCCTCCC TCCTCCTCCT CGTCGACGAA
     2341 GGTCTCGATC TCCATGTTCT CGCCGGCCAC GTTGATGGTG GTTGTCTTCC GGATGTCGCC
     2401 GTGGATCAGG ATGTGCTCGT GGATCTTCAC TCTCAGGTGG TGGCCCAGGA AGCCCCAAGT
     2461 GTTCTTGCTG ATCAGATCCA GGATCAGGTG GCCGATTCCG CTGGTGTAAG GTGTGATCAG
     2521 GCCCAGTCCC TCGTGTCCGG CGATGAACAT CACGGGGAAC ATCAGCCAAG AGCAGAAGAA
     2581 GACGTAGGCC ATGATCTTGC AAATCTTCCG GCAGACGCCT TTGGGCAGGG TGTGGTAGCT
     2641 CTCGATATAC ACCTTGGCGA TCTGGAAGAA GCAAGTCACG CCGTAGAACA GGCCGATCAT
     2701 GAAGAACAGG ATCTTCAGGG GGCCCTTTGT AAAGGCGGCT GTGATCCCCC ACACGATGTT
     2761 GCCGATGTCG GTCACCAGGA TGGTCATGGT CCGCTTGCTG TACTCTTCGT GCAGTCCGGT
     2821 CAGGTTGCTC AGGTGGATCA GGATCACGGG GCAAGTCAGG AGCCACATGC TGTACCGCAG
     2881 CCAAATCACG GCTCCTCCGT TGGTCTGGTA CACTGTGGCT GGGCTGTCGA CCTCGTGGAA
     2941 CAGCTCGATG AAGCACTTGA CGAGCTCGAT CACGCAGACG TAGACCTCCT CCCAGCCGGT
     3001 TGTGGCCTTG AAGCTGTGCC AACCGTAGAA GATGAGCTGC ACGATAGCCA CGATCACGGT
     3061 GAACCACTGG AGTCCCACTG CGATCTTGTG CTGCAGCTCG GTTCCGTGGT TGATGTGGGG
     3121 GAAGCAACCG TGATCGGCTC CGGCTGTTGT GGCGTTAGAG GTTTCTCCGT GAGCGTCTGC
     3181 TGCTGGTGTC ACCACTGCTG CGGCGCTCAG CAGGCCTCTA ATTGTGGCTT CGGCGCTAGG
     3241 CACGGCTGAG ATAAAGGCGT GGGTCATTGT GGCGGCTGTT TCCATggtgg cgctagcgaa
     3301 gttcctatac tttctagaga ataggaactt cttgccttaa cccagaaatt atcactgtta
     3361 ttctttagaa tggtgcaaag agaagttcct ataccttttg aagaatagga acttcgaatt
     3421 cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg
     3481 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     3541 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     3601 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     3661 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     3721 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     3781 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     3841 gtcctttcct tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg
     3901 ctacgtccct tcggcccCCA AGGAAAGGAC GATGATTTCt caatccagcg gaccttcctc
     3961 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     4021 gtcggatctc cctttgggcc gcctccccgc atcgataccg agcgctgctc gagagatcta
     4081 cgggtggcat ccctgtgacc cctccccagt gcctctcctg gccctggaag ttgccactcc
     4141 agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg actaggtgtc
     4201 cttctataat attatggggt ggaggggggt ggtatggagc aaggggcaag ttgggaagac
     4261 aacctgtagg gcctgcgggg tctattggga accaagctgg agtgcagtgg cacaatcttg
     4321 gctcactgca atctccgcct cctgggttca agcgattctc ctgcctcagc ctcccgagtt
     4381 gttgggattc caggcatgca tgaccaggct cagctaattt ttgttttttt ggtagagacg
     4441 gggtttcacc atattggcca ggctggtctc caactcctaa tctcaggtga tctacccacc
     4501 ttggcctccc aaattgctgg gattacaggc gtgaaccact gctcccttcc ctgtccttct
     4561 gattttgtag gtaaccacgt gcggaccgag cggccgcagg aacccctagt gatggagttg
     4621 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     4681 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     4741 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     4801 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4861 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     4921 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4981 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     5041 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     5101 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcgggctatt
     5161 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     5221 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     5281 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     5341 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     5401 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     5461 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     5521 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5581 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5641 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5701 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5761 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5821 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5881 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5941 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     6001 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     6061 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     6121 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     6181 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     6241 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6301 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6361 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6421 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6481 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6541 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6601 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6661 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6721 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6781 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     6841 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6901 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6961 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     7021 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     7081 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     7141 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     7201 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     7261 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     7321 cttttgctca catgt
//