LOCUS       sequence-182997-        6677 bp    DNA     circular SYN 14-APR-2025
DEFINITION  AAV mediated Cre-dependent expression of EGFP (Lox-Stop-Lox) .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6677)
  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 6677)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/nature13186"; journalName: "Nature"; date: "2014-04-10- 
            10"; volume: "508"; issue: "7495"; pages: "207-14"
COMMENT     Sequence Label: pAAV.CAG.LSL.EGFP
FEATURES             Location/Qualifiers
     source          1..6677
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     enhancer        234..537
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        539..816
                     /label=chicken beta-actin promoter
     intron          817..1833
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1757..1779
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1841..1860
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(1961..1994)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     polyA_signal    2162..2296
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    2416..2550
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    2670..2804
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(2833..2866)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     regulatory      2901..2910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2907..2909,2910..2912,2913..3626)
                     /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             join(2907..2909,2910..2912,2913..3623)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2952..2973)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3213..3232)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3560..3581
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3704..3721)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3710..3917
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3940..4080
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3940..4069
                     /label=AAV2 ITR
     rep_origin      4155..4610
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4242..4261)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4452..4473
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4627..4646)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4746..4768
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4806..4824)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4892..4996
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4997..5065,5066..5857)
                     /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(5215..5234)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6028..6616
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6517..6536
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgaaacaa ttctgcagga atctagttat
      181 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
      241 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      301 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      361 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      421 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
      481 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc
      541 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
      601 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg
      661 cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg
      721 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      781 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc
      841 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
      901 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gcgcttggtt
      961 taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct ccgggagggc
     1021 cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt ggggagcgcc
     1081 gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt
     1141 gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg gtgcgggggg
     1201 ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg agcagggggt
     1261 gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc ctccccgagt tgctgagcac
     1321 ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg cggggctcgc cgtgccgggc
     1381 ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc cggggagggc
     1441 tcgggggagg ggcgcggcgg cccccggagc gccggcggct gtcgaggcgc ggcgagccgc
     1501 agccattgcc ttttatggta atcgtgcgag agggcgcagg gacttccttt gtcccaaatc
     1561 tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg cggggcgaag
     1621 cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc gccgcgccgc
     1681 cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg ccttcggggg
     1741 ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag agcctctgct
     1801 aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct ggttattgtg
     1861 ctgtctcatc attttggcaa agaattgatt tgataccgcg ggccccgcgt ggacggtacc
     1921 cttcaggtcg acatttatcc ctaaacagga taacactagc ataacttcgt ataggatact
     1981 ttatacgaag ttattcgcga tgaataaatg aaagcttgca gatctgcgac tctagaggat
     2041 ctgcgactct agaggatcat aatcagccat accacatttt gtagaggttt tacttgcttt
     2101 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     2161 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     2221 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     2281 ttatcatgtc tggatctgcg actctagagg atcataatca gccataccac atttgtagag
     2341 gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat
     2401 gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     2461 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     2521 ctcatcaatg tatcttatca tgtctggatc tgcgactcta gaggatcata atcagccata
     2581 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     2641 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     2701 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     2761 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatccccatc aagctgatcc
     2821 ggaaccctta atataacttc gtataggata ctttatacga agttattagc tagttacagt
     2881 accggcttag ttaggaattc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg
     2941 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     3001 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     3061 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     3121 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     3181 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     3241 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     3301 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     3361 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     3421 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     3481 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     3541 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     3601 tcggcatgga cgagctgtac aagtaagaat tctaaatttc gaaagtcgac gtcggttggc
     3661 gcgccaaggg tttaaacccc ctggccggcc ttgcgaagat ctgcctcgac tgtgccttct
     3721 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     3781 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     3841 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     3901 agcaggcatg ctggggacac gtgcggaccg agcggccgca ggaaccccta gtgatggagt
     3961 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     4021 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     4081 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     4141 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4201 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     4261 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     4321 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     4381 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     4441 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta
     4501 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     4561 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     4621 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     4681 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4741 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     4801 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     4861 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     4921 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     4981 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     5041 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     5101 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     5161 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     5221 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     5281 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     5341 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     5401 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     5461 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     5521 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     5581 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     5641 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     5701 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     5761 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     5821 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     5881 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     5941 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     6001 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     6061 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     6121 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     6181 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     6241 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     6301 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     6361 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     6421 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     6481 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     6541 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     6601 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     6661 gccttttgct cacatgt
//
