LOCUS       sequence-407140-        6558 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Can be used to generate AAV virus that will express EGFP in the 
            presence of Cre.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6558)
  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 6558)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 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: AAV pCAG-FLEX-EGFP-WPRE
FEATURES             Location/Qualifiers
     source          1..6558
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..780
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        873..1176
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1178..1460
                     /label=chicken beta-actin promoter
     intron          1461..2477
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2401..2423
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2485..2504
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(2581..2614)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(2625..2658)
                     /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)."
     CDS             complement(join(2714..3427,3428..3430,3431..3433))
                     /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(join(2717..3427,3428..3430,3431..3433))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2759..2780)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3108..3127
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3367..3388
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3430..3439
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3511..3544
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3555..3588
                     /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)."
     misc_feature    3612..4200
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3665..3685)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4083..4094)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4235..4252)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4241..4448
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4471..4611
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4471..4600
                     /label=AAV2 ITR
     rep_origin      4686..5141
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4773..4792)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4983..5004
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5158..5177)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5277..5299
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5337..5355)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5423..5527
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5528..5596,5597..6388)
                     /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(5746..5765)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
      721 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      781 gcggccgcac gcgaaacaat tctgcaggaa tctagttatt aatagtaatc aattacgggg
      841 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      901 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      961 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     1021 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     1081 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     1141 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
     1201 ttcactctcc ccatctcccc ccccctcccc acccccaatt ttgtatttat ttatttttta
     1261 attattttgt gcagcgatgg gggcgggggg gggggggggg ggggcgcgcg ccaggcgggg
     1321 cggggcgggg cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga
     1381 gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa
     1441 agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg
     1501 ccgccgcctc gcgccgcccg ccccggctct gactgaccgc gttactccca caggtgagcg
     1561 ggcgggacgg cccttctcct ccgggctgta attagcgctt ggtttaatga cggcttgttt
     1621 cttttctgtg gctgcgtgaa agccttgagg ggctccggga gggccctttg tgcgggggga
     1681 gcggctcggg gggtgcgtgc gtgtgtgtgt gcgtggggag cgccgcgtgc ggctccgcgc
     1741 tgcccggcgg ctgtgagcgc tgcgggcgcg gcgcggggct ttgtgcgctc cgcagtgtgc
     1801 gcgaggggag cgcggccggg ggcggtgccc cgcggtgcgg ggggggctgc gaggggaaca
     1861 aaggctgcgt gcggggtgtg tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg
     1921 ggctgcaacc ccccctgcac ccccctcccc gagttgctga gcacggcccg gcttcgggtg
     1981 cggggctccg tacggggcgt ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg
     2041 tgggggtgcc gggcggggcg gggccgcctc gggccgggga gggctcgggg gaggggcgcg
     2101 gcggcccccg gagcgccggc ggctgtcgag gcgcggcgag ccgcagccat tgccttttat
     2161 ggtaatcgtg cgagagggcg cagggacttc ctttgtccca aatctgtgcg gagccgaaat
     2221 ctgggaggcg ccgccgcacc ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca
     2281 ggaaggaaat gggcggggag ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc
     2341 tccagcctcg gggctgtccg cggggggacg gctgccttcg ggggggacgg ggcagggcgg
     2401 ggttcggctt ctggcgtgtg accggcggct ctagagcctc tgctaaccat gttcatgcct
     2461 tcttcttttt cctacagctc ctgggcaacg tgctggttat tgtgctgtct catcattttg
     2521 gcaaagaatt gatttgatac cgcgggcccc gcgtggacgg tacccttcag gtcgacgtcg
     2581 ataacttcgt atagcataca ttatacgaag ttatccgaat cgcaataact tcgtatagga
     2641 tactttatac gaagttattt ggcgcgccaa atttaaataa cgcaacgtac cttcgagggg
     2701 gcccaaggaa ttcttacttg tacagctcgt ccatgccgag agtgatcccg gcggcggtca
     2761 cgaactccag caggaccatg tgatcgcgct tctcgttggg gtctttgctc agggcggact
     2821 gggtgctcag gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct
     2881 gctggtagtg gtcggcgagc tgcacgctgc cgtcctcgat gttgtggcgg atcttgaagt
     2941 tcaccttgat gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg ctgttgtagt
     3001 tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg cccttcagct
     3061 cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc
     3121 cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga
     3181 agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg ccgtaggtca
     3241 gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag atgaacttca
     3301 gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg aacttgtggc
     3361 cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac agctcctcgc
     3421 ccttgctcac catggtggcg aattccccca agcttggaac gtactaacct tgatagctag
     3481 ctagttggcg cgccaacctg gccggccttg ataacttcgt ataatgtatg ctatacgaag
     3541 ttatccgaat cgcaataact tcgtataaag tatcctatac gaagttatcg aagatcatca
     3601 agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     3661 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     3721 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     3781 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     3841 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     3901 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     3961 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     4021 cttggctgct cgcctatgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     4081 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     4141 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     4201 atcgataccg agcgctgctc gagagatcga tctgcctcga ctgtgccttc tagttgccag
     4261 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     4321 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     4381 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     4441 gctggggaca cgtgcggacc gagcggccgc aggaacccct agtgatggag ttggccactc
     4501 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     4561 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga
     4621 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc
     4681 atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     4741 gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct
     4801 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     4861 atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag
     4921 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     4981 tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga
     5041 tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa
     5101 atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac
     5161 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     5221 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     5281 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
     5341 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     5401 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5461 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5521 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     5581 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5641 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     5701 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     5761 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     5821 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     5881 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     5941 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     6001 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     6061 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     6121 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     6181 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     6241 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     6301 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     6361 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     6421 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     6481 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     6541 aaagatcaaa ggatcttc
//
