LOCUS       sequence-486471-        5651 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV transfer plasmid expressing eGFP-E2 under a CAG promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5651)
  AUTHORS   Park SY, Sheridan A, An B, Jarvis E, Lyudchik J, Patton W, Axup JY, 
            Chan SW, Damstra HGJ, Leible D, Leung KS, Magno CA, Meeran A, 
            Michalska JM, Rieger F, Wang C, Wu M, Church GM, Funke J, Huffman T,
            Leeper KGC, Truckenbrodt S, Winnubst J, Kornfeld JMR, Boyden ES, 
            Rodriques SG, Payne AC
  TITLE     Combinatorial protein barcodes enable self-correcting neuron tracing
            with nanoscale molecular context
  JOURNAL   bioRxiv 2025.09.26.678648
REFERENCE   2  (bases 1 to 5651)
  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: "bioRxiv 
            2025.09.26.678648"
COMMENT     Sequence Label: pAAV-CAG-eGFP-E2
FEATURES             Location/Qualifiers
     source          1..5651
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..30
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(249..353)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(379..834)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(516..537)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     728..747
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   906..1035
                     /label=AAV2 ITR
     promoter        1346..1621
                     /label=chicken beta-actin promoter
     primer_bind     1900..1922
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1984..2003
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2036..2045
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2042..2044,2045..2047,2048..2758)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2042..2044,2045..2047,2048..2758)
                     /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(2087..2108)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2348..2367)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2695..2716
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3000..3588
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3053..3073)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3471..3482)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3631..3752
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3642..3661)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3696..3715
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3914..4043
                     /label=AAV2 ITR
     rep_origin      complement(4280..4868)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4360..4379)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5039..5651,1..179,180..248))
                     /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"
ORIGIN
        1 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
       61 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
      121 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
      181 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
      241 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
      301 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
      361 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
      421 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
      481 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
      541 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
      601 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
      661 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
      721 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
      781 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
      841 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
      901 gccagctgcg cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga
      961 cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca
     1021 tcactagggg ttccttgtag ttaatgatta acccgccatg ctacttatct acgtagccat
     1081 gctctaggaa gagtaccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     1141 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     1201 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     1261 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     1321 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
     1381 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
     1441 gcgatggggg cggggggggg gggggsgcgc gccaggcggg gcggggcggg gcgaggggcg
     1501 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
     1561 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
     1621 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
     1681 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
     1741 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
     1801 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggctgtccgc
     1861 ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga
     1921 ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc
     1981 tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattg gatccgccac
     2041 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     2101 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     2161 cggcaagctg accctgaagt tcatttgcac caccggcaag ctgcccgtgc cctggcccac
     2221 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     2281 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     2341 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     2401 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     2461 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     2521 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     2581 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     2641 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     2701 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggg
     2761 tggatctggt ggcagttcat caactagctc cgattttcgg gatagagggg gctcaggtgg
     2821 ctcttcctcc actagtagtg actttagaga caggggagga agcggagggt cctctagcac
     2881 cagttctgac ttccgagatc gctaagaatt cttcagtagg actggttatg aggacgaatc
     2941 tcccgcttat accaggtcaa gcttatcgat aatcagaatt cgatatcaag cttatcgata
     3001 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     3061 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     3121 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     3181 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     3241 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     3301 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     3361 tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg
     3421 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     3481 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     3541 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc
     3601 gacccgggcg gccgcttcga gcagacatga taagatacat tgatgagttt ggacaaacca
     3661 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     3721 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     3781 ttcaggttca gggggagatg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
     3841 gtaaaatcga taaggatctt cctagagcat ggctacgtag ataagtagca tggcgggtta
     3901 atcattaact acaaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     3961 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     4021 tcagtgagcg agcgagcgcg cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4081 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4141 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4201 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4261 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4321 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4381 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4441 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4501 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4561 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4621 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4681 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4741 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4801 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4861 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4921 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4981 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5041 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5101 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5161 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5221 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5281 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5341 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5401 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5461 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5521 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5581 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5641 cttgcccggc g
//
