LOCUS       sequence-92028-e        6951 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses splitTVA-P2A-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6951)
  TITLE     Genetic Neuroengineering Group Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6951)
  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            "Unpublished"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-splitTVA-EGFP
FEATURES             Location/Qualifiers
     source          1..6951
                     /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"
     regulatory      1873..1882
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1888..1921
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1972..2005
                     /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(2014..2727,2728..2730,2731..2733))
                     /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(2017..2727,2728..2730,2731..2733))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2059..2080)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2408..2427
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2667..2688
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2734..2790)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     protein_bind    complement(3273..3306)
                     /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(3357..3390)
                     /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    3392..3980
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3445..3465)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3863..3874)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3988..4212
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4214..4354
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4214..4343
                     /label=AAV2 ITR
     rep_origin      4429..4884
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4516..4535)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4726..4747
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4901..4920)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5020..5042
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5080..5098)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5166..5270
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5271..5339,5340..6131)
                     /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(5489..5508)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6302..6890
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6791..6810
                     /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 aattccggag ccgccaccat ggttaatata acttcgtata atgtatgcta tacgaagtta
     1921 tgcagaatgg tagctggatt gtagctgcta ttagcaatat gaaacctctt aataacttcg
     1981 tataaagtat cctatacgaa gttatggcgc gccttacttg tacagctcgt ccatgccgag
     2041 agtgatcccg gcggcggtca cgaactccag caggaccatg tgatcgcgct tctcgttggg
     2101 gtctttgctc agggcggact gggtgctcag gtagtggttg tcgggcagca gcacggggcc
     2161 gtcgccgatg ggggtgttct gctggtagtg gtcggcgagc tgcacgctgc cgtcctcgat
     2221 gttgtggcgg atcttgaagt tcaccttgat gccgttcttc tgcttgtcgg ccatgatata
     2281 gacgttgtgg ctgttgtagt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt
     2341 gaagtcgatg cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc
     2401 ggcgcgggtc ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc
     2461 ttcgggcatg gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa
     2521 gcactgcacg ccgtaggtca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc
     2581 ggtggtgcag atgaacttca gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc
     2641 ggacacgctg aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac
     2701 cccggtgaac agctcctcgc ccttgctcac catgggtcca gggttctcct ccacgtctcc
     2761 agcctgcttc agcaggctga agttagtagc tccggatccg gagaacaagt ctgcctggct
     2821 cttgtcaggc accagcagct ccttggatgc gctttcaaga ctgaagatgt cagacctgct
     2881 ttttgctttg gacttccccc atgcagcgat accacccaca gctaccaggc agcacaggag
     2941 cactgcagtg atcagcatcc acatgcggcc gtgattcctg gctggcagag cacgtccagg
     3001 agcaggggca gtgggagcct ctgtgccgtt gtccgtgggc accgcgggga tcgcgctggt
     3061 cccgcagccc cactcgtccc gcccgtcgtc gcagtcgggg tgtccgtcgc acagccagtc
     3121 ctgcgggtaa cactccccgt gggcaccggg cggctccgag cagcggaact gaccgggggg
     3181 gcaacgggac aaagaaccgt taccggaccc gttaccggtc acgttaccgg gcagcagcag
     3241 cagcagcagc gcgggcagca gccgcgcacc taataacttc gtatagcata cattatacga
     3301 agttatttgc cttaacccag aaattatcac tgttattctt tagaatggtg caaagaataa
     3361 cttcgtatag gatactttat acgaagttat gaatcaacct ctggattaca aaatttgtga
     3421 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     3481 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     3541 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     3601 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     3661 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     3721 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     3781 ggggaagctg acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     3841 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     3901 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     3961 cctttgggcc gcctccccgc ctggatcctg tgccttctag ttgccagcca tctgttgttt
     4021 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     4081 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     4141 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     4201 tgggctctat ggcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     4261 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     4321 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     4381 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     4441 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4501 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     4561 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4621 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     4681 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4741 aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc
     4801 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     4861 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     4921 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     4981 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     5041 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     5101 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     5161 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5221 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     5281 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5341 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5401 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5461 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5521 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5581 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5641 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     5701 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     5761 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     5821 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     5881 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     5941 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     6001 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6061 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     6121 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     6181 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6241 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6301 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6361 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6421 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     6481 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6541 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6601 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6661 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6721 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6781 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     6841 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     6901 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg t
//
