LOCUS       sequence-92029-e        6795 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP-P2A-B19G.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6795)
  TITLE     Genetic Neuroengineering Group Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6795)
  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            "Unpublished"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-synP-FLEX-EGFP-B19G
FEATURES             Location/Qualifiers
     source          1..6795
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        164..611
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(620..653)
                     /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(704..737)
                     /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(2319..2375)
                     /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"
     CDS             complement(join(2385..3095,3096..3098,3099..3101))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(2385..3095,3096..3098,3099..3101))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2427..2448)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2776..2795
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3035..3056
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3098..3107
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3113..3146
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3197..3230
                     /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    3236..3824
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3289..3309)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3707..3718)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3832..4056
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4058..4198
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4058..4187
                     /label=AAV2 ITR
     rep_origin      4273..4728
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4360..4379)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4570..4591
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4745..4764)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4864..4886
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4924..4942)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5010..5114
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5115..5183,5184..5975)
                     /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(5333..5352)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6146..6734
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6635..6654
                     /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 gtcgacgcta gactgcagag ggccctgcgt atgagtgcaa gtgggtttta
      181 ggaccaggat gaggcggggt gggggtgcct acctgacgac cgaccccgac ccactggaca
      241 agcacccaac ccccattccc caaattgcgc atcccctatc agagaggggg aggggaaaca
      301 ggatgcggcg aggcgcgtgc gcactgccag cttcagcacc gcggacagtg ccttcgcccc
      361 cgcctggcgg cgcgcgccac cgccgcctca gcactgaagg cgcgctgacg tcactcgccg
      421 gtcccccgca aactcccctt cccggccacc ttggtcgcgt ccgcgccgcc gccggcccag
      481 ccggaccgca ccacgcgagg cgcgagatag gggggcacgg gcgcgaccat ctgcgctgcg
      541 gcgccggcga ctcagcgctg cctcagtctg cggtgggcag cggaggagtc gtgtcgtgcc
      601 tgagagcgca gtcgaattca taacttcgta tagcatacat tatacgaagt tattctttgc
      661 accattctaa agaataacag tgataatttc tgggttaagg caaataactt cgtataggat
      721 actttatacg aagttatgct agcttacagt ctggtctcac ccccactctt gtgtgattcc
      781 catgaagata tgatcttccc gctttgggga gtgactgaca cctccctccc tgtccctctg
      841 agattgtgtt gcgtaggttc tgatcgattg actcttctac aacatgtcat caggaaaatt
      901 atcaacatca aggcagtcag ggcccctgca ctcagtaata catacttccc ccagttcggg
      961 agacccaagt caactcctga gacctgattg tgcacatcgg gaaggtgaac ttcaacaaaa
     1021 tcctcagcct cgtcaccgtc cttgaaaacg gtagacgggt ctgccagggg gtgcacaagg
     1081 gggataaccg aggattccaa caactccata tgttgctgga ggagggatga ttgcatctct
     1141 gggattaaga cattgccgtc aggtcctaat attataccat tgaaaaacac cccgttcaca
     1201 tgaggatgac acctcccccc aactcttaaa cacccttttg aagggaggat ctcattccaa
     1261 gttctgactg acttgtagtg agcatcggct tccatcaagg tcttgttgaa tatggtatat
     1321 gcttttccaa acccagggac aagttttctt aaatgactga gacgtctgaa actcactgac
     1381 ttggttgtca tgatggactc tagtgcatcc agacactcct ctctcttcct gaccaactcc
     1441 tctacaacaa ggtgctcaat ttcgtctgag cgaaagtcgt gcaggttcac caacttatcg
     1501 ggagggcacc atttggtttc atttgatgtt tgcatcgaga cccatgttcc atccataagt
     1561 ctaagtccta gaactccaca taacttgagt ttgcatgctc cttttaaaga cttatatagg
     1621 cctctttcat ctacaaagcc gcaagtctca ctccctttgg atgctctctt ccctctacta
     1681 ttggtaaaaa tgtcacaaga catccctagt ctcggattct cgggcatcca aatggtgtaa
     1741 tcgtggttag tggagcagta ggtagaagac accgctactc ctgagcactt cccgctaggg
     1801 aagaccctcg agtgaaggga tctgtcatat gggtccaaat ctgccacact tggagatatg
     1861 ataacgagag actccttggt ggtttttaca gttcgaagcc agcggtagtc agggtacgga
     1921 ttgtgtagag actcttcata tctggggtca ccggccatct tccagttgta cgcggctcta
     1981 catgcatctg gtgttgggcg gaaatgcttt cttttgaacg tggttgtgac ataaccaacg
     2041 aagttagtgt aggtttcagc ctccgtcaca acgcctgtgc aagtgaaccc gttcactttt
     2101 atggctaaga tgtatccaac tttaagttcc atgtaggaga accctgacag gttggtgcat
     2161 ccttcgtcct ccactaccaa attgtttggg cagctgaggt gatgtatgtc aatcggactc
     2221 cagggaccaa gcttgtctgg tatcgtgtaa atagggaatt tcccaaaaca caatggaaaa
     2281 accagaaggg gtacaaacag gagagcctga ggaaccatgg gtccagggtt ctcctccacg
     2341 tctccagcct gcttcagcag gctgaagtta gtagctccgg atcccttgta cagctcgtcc
     2401 atgccgagag tgatcccggc ggcggtcacg aactccagca ggaccatgtg atcgcgcttc
     2461 tcgttggggt ctttgctcag ggcggactgg gtgctcaggt agtggttgtc gggcagcagc
     2521 acggggccgt cgccgatggg ggtgttctgc tggtagtggt cggcgagctg cacgctgccg
     2581 tcctcgatgt tgtggcggat cttgaagttc accttgatgc cgttcttctg cttgtcggcc
     2641 atgatataga cgttgtggct gttgtagttg tactccagct tgtgccccag gatgttgccg
     2701 tcctccttga agtcgatgcc cttcagctcg atgcggttca ccagggtgtc gccctcgaac
     2761 ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga agaagatggt gcgctcctgg
     2821 acgtagcctt cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag
     2881 cggctgaagc actgcacgcc gtaggtcagg gtggtcacga gggtgggcca gggcacgggc
     2941 agcttgccgg tggtgcagat gaacttcagg gtcagcttgc cgtaggtggc atcgccctcg
     3001 ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc cgtccagctc gaccaggatg
     3061 ggcaccaccc cggtgaacag ctcctcgccc ttgctcacca tggtggcgcg ccataacttc
     3121 gtataatgta tgctatacga agttattaag aggtttcata ttgctaatag cagctacaat
     3181 ccagctacca ttctgcataa cttcgtataa agtatcctat acgaagttat tccggaatca
     3241 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     3301 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     3361 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
     3421 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
     3481 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
     3541 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
     3601 cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg
     3661 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
     3721 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
     3781 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcctggat cctgtgcctt
     3841 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     3901 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     3961 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     4021 atagcaggca tgctggggat gcggtgggct ctatggcagg aacccctagt gatggagttg
     4081 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     4141 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     4201 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     4261 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4321 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     4381 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4441 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     4501 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4561 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcgggctatt
     4621 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     4681 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     4741 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     4801 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4861 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     4921 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     4981 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5041 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5101 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5161 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5221 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5281 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5341 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5401 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5461 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5521 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5581 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5641 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     5701 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     5761 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5821 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5881 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5941 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6001 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6061 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6121 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6181 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6241 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     6301 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6361 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6421 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6481 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6541 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6601 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6661 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6721 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6781 cttttgctca catgt
//
