LOCUS       sequence-107230-        6841 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TVA66T-EGFP under the CAG promotor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6841)
  TITLE     Genetic Neuroengineering Group Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6841)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-CAG-FLEX-TVA66T-EGFP
FEATURES             Location/Qualifiers
     source          1..6841
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        134..513
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        515..792
                     /label=chicken beta-actin promoter
     intron          794..1811
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1735..1757
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1819..1838
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(1866..1899)
                     /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(1950..1983)
                     /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(1990..2703,2704..2706))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(1993..2703,2704..2706))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2035..2056)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2384..2403
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2643..2664
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3141..3150
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3159..3192
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3243..3276
                     /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    3282..3870
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3335..3355)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3753..3764)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3878..4102
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4104..4244
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4104..4233
                     /label=AAV2 ITR
     rep_origin      4319..4774
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4406..4425)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4616..4637
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4791..4810)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4910..4932
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4970..4988)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5056..5160
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5161..5229,5230..6021)
                     /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(5379..5398)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6192..6780
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6681..6700
                     /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 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca
      181 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      241 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      301 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      361 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      421 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      481 tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc
      541 actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta
      601 ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg
      661 ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg
      721 ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc
      781 gcgcggcggg cggggagtcg ctgcgacgct gccttcgccc cgtgccccgc tccgccgccg
      841 cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg
      901 acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc
      961 tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct
     1021 cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg
     1081 gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg
     1141 ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct
     1201 gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc
     1261 aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc
     1321 tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggtggggg
     1381 tgccgggcgg ggcggggccg cctcgggccg gggagggctc gggggagggg cgcggcggcc
     1441 cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat
     1501 cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga
     1561 ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg
     1621 aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc
     1681 ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg
     1741 gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct
     1801 ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat tttggcaaag
     1861 aattcataac ttcgtatagc atacattata cgaagttatt ctttgcacca ttctaaagaa
     1921 taacagtgat aatttctggg ttaaggcaaa taacttcgta taggatactt tatacgaagt
     1981 tatgctagct cacttgtaca gctcgtccat gccgagagtg atcccggcgg cggtcacgaa
     2041 ctccagcagg accatgtgat cgcgcttctc gttggggtct ttgctcaggg cggactgggt
     2101 gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg tgttctgctg
     2161 gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg tggcggatct tgaagttcac
     2221 cttgatgccg ttcttctgct tgtcggccat gatatagacg ttgtggctgt tgtagttgta
     2281 ctccagcttg tgccccagga tgttgccgtc ctccttgaag tcgatgccct tcagctcgat
     2341 gcggttcacc agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt agttgccgtc
     2401 gtccttgaag aagatggtgc gctcctggac gtagccttcg ggcatggcgg acttgaagaa
     2461 gtcgtgctgc ttcatgtggt cggggtagcg gctgaagcac tgcacgccgt aggtcagggt
     2521 ggtcacgagg gtgggccagg gcacgggcag cttgccggtg gtgcagatga acttcagggt
     2581 cagcttgccg taggtggcat cgccctcgcc ctcgccggac acgctgaact tgtggccgtt
     2641 tacgtcgccg tccagctcga ccaggatggg caccaccccg gtgaacagct cctcgccctt
     2701 gctcacccca ccccccttgg atgcgctttc aagactgaag atgtcagacc tgctttttgc
     2761 tttggacttc ccccatgcag cgataccacc cacagctacc aggcagcaca ggagcactgc
     2821 agtgatcagc atccacatgc ggccgtgatt cctggctggc agagcacgtc caggagcagg
     2881 ggcagtggga gcctctgtgc cgttgtccgt gggcaccgcg gggatcgcgc tggtcccgca
     2941 gccccaggtg tcccgcccgt cgtcgcagtc ggggtgtccg tcgcacagcc agtcctgcgg
     3001 gtaacactcc ccgtgggcac cgggcggctc cgagcagcgg aactgaccgg gggggcaacg
     3061 ggacaaagaa ccgttaccgg acccgttacc ggtcacgtta ccgggcagca gcagcagcag
     3121 cagcgcgggc agcagccgcg ccatggtggc ggcgcgccat aacttcgtat aatgtatgct
     3181 atacgaagtt attaagaggt ttcatattgc taatagcagc tacaatccag ctaccattct
     3241 gcataacttc gtataaagta tcctatacga agttattccg gaatcaacct ctggattaca
     3301 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     3361 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     3421 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     3481 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     3541 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     3601 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     3661 tggtgttgtc ggggaagctg acgtcctttc catggctgct cgcctgtgtt gccacctgga
     3721 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     3781 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     3841 gtcggatctc cctttgggcc gcctccccgc ctggatcctg tgccttctag ttgccagcca
     3901 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     3961 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     4021 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     4081 ggggatgcgg tgggctctat ggcaggaacc cctagtgatg gagttggcca ctccctctct
     4141 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     4201 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta
     4261 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac
     4321 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     4381 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     4441 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     4501 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca
     4561 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4621 ctcttgttcc aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa
     4681 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4741 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc
     4801 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     4861 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4921 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     4981 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     5041 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5101 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5161 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5221 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5281 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5341 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5401 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5461 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5521 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5581 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5641 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5701 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5761 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     5821 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     5881 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     5941 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6001 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6061 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     6121 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6181 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     6241 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6301 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     6361 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6421 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6481 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6541 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6601 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6661 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6721 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6781 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     6841 t
//
