LOCUS       Exported                6555 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Can be used to generate AAV virus that will express EGFP in the 
            presence of Cre.
ACCESSION   .
VERSION     .
KEYWORDS    AAV pCAG-FLEX-EGFP-WPRE
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6555)
  AUTHORS   Oh SW, Harris JA, Ng L, Winslow B, Cain N, Mihalas S, Wang Q, Lau C,
            Kuan L, Henry AM, Mortrud MT, Ouellette B, Nguyen TN, Sorensen SA, 
            Slaughterbeck CR, Wakeman W, Li Y, Feng D, Ho A, Nicholas E, 
            Hirokawa KE, Bohn P, Joines KM, Peng H, Hawrylycz MJ, Phillips JW, 
            Hohmann JG, Wohnoutka P, Gerfen CR, Koch C, Bernard A, Dang C, Jones
            AR, Zeng H
  TITLE     A mesoscale connectome of the mouse brain.
  JOURNAL   Nature. 2014 Apr 10;508(7495):207-14. doi: 10.1038/nature13186. Epub
            2014 Apr 2.
  PUBMED    24695228
REFERENCE   2  (bases 1 to 6555)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6555
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        223..526
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        528..805
                     /label=chicken beta-actin promoter
     intron          807..1824
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1748..1770
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1832..1851
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1928..1961
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    1972..2005
                     /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)."
     CDS             complement(2061..2780)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(2064..2780)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2106..2127)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2455..2474
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2714..2735
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2777..2786
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(2858..2891)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    complement(2902..2935)
                     /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)."
     misc_feature    2959..3547
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3012..3032)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3430..3441)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3582..3599)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3588..3795
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3818..3958
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3818..3947
                     /label=AAV2 ITR
     rep_origin      4033..4488
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4120..4139)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4330..4351
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4505..4524)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4624..4646
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4684..4702)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4770..4874
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4875..5735
                     /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(5093..5112)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5906..6494
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6395..6414
                     /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 gcgaaacaat tctgcaggaa tctagttatt aatagtaatc
      181 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      241 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      301 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      361 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      421 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      481 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
      541 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
      601 tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggc gcgcgccagg
      661 cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa
      721 tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta
      781 taaaaagcga agcgcgcggc gggcggggag tcgctgcgac gctgccttcg ccccgtgccc
      841 cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag
      901 gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg
      961 cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc
     1021 ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc
     1081 tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc
     1141 agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag
     1201 gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg
     1261 tcggtcgggc tgcaaccccc cctgcacccc cctccccgag ttgctgagca cggcccggct
     1321 tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg
     1381 cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg ccggggaggg ctcgggggag
     1441 gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc
     1501 cttttatggt aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag
     1561 ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc
     1621 gccggcagga aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt
     1681 ctccctctcc agcctcgggg ctgtccgcgg ggggacggct gccttcgggg gggacggggc
     1741 agggcggggt tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt
     1801 catgccttct tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat
     1861 cattttggca aagaattgat ttgataccgc gggccccgcg tggacggtac ccttcaggtc
     1921 gacgtcgata acttcgtata gcatacatta tacgaagtta tccgaatcgc aataacttcg
     1981 tataggatac tttatacgaa gttatttggc gcgccaaatt taaataacgc aacgtacctt
     2041 cgagggggcc caaggaattc ttacttgtac agctcgtcca tgccgagagt gatcccggcg
     2101 gcggtcacga actccagcag gaccatgtga tcgcgcttct cgttggggtc tttgctcagg
     2161 gcggactggg tgctcaggta gtggttgtcg ggcagcagca cggggccgtc gccgatgggg
     2221 gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt cctcgatgtt gtggcggatc
     2281 ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca tgatatagac gttgtggctg
     2341 ttgtagttgt actccagctt gtgccccagg atgttgccgt cctccttgaa gtcgatgccc
     2401 ttcagctcga tgcggttcac cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg
     2461 tagttgccgt cgtccttgaa gaagatggtg cgctcctgga cgtagccttc gggcatggcg
     2521 gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg
     2581 taggtcaggg tggtcacgag ggtgggccag ggcacgggca gcttgccggt ggtgcagatg
     2641 aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga cacgctgaac
     2701 ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg gcaccacccc ggtgaacagc
     2761 tcctcgccct tgctcaccat ggtggcgaat tcccccaagc ttggaacgta ctaaccttga
     2821 tagctagcta gttggcgcgc caacctggcc ggccttgata acttcgtata atgtatgcta
     2881 tacgaagtta tccgaatcgc aataacttcg tataaagtat cctatacgaa gttatcgaag
     2941 atcatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     3001 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     3061 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     3121 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     3181 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     3241 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     3301 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
     3361 tcctttcctt ggctgctcgc ctatgttgcc acctggattc tgcgcgggac gtccttctgc
     3421 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     3481 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     3541 tccccgcatc gataccgagc gctgctcgag agatcgatct gcctcgactg tgccttctag
     3601 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     3661 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     3721 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     3781 caggcatgct ggggacacgt gcggaccgag cggccgcagg aacccctagt gatggagttg
     3841 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     3901 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     3961 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     4021 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4081 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
     4141 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4201 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     4261 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4321 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
     4381 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
     4441 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     4501 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     4561 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4621 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     4681 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     4741 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4801 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4861 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4921 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     4981 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5041 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5101 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5161 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5221 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5281 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5341 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5401 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     5461 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     5521 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5581 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5641 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5701 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5761 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5821 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5881 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5941 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6001 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6061 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6121 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6181 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6241 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6301 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6361 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6421 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6481 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6541 cttttgctca catgt
//
