LOCUS       sequence-486470-        5547 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV transfer plasmid expressing eGFP-AU5 under a CAG promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5547)
  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 5547)
  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-AU5
FEATURES             Location/Qualifiers
     source          1..5547
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     14..33
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(252..356)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(382..837)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(519..540)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     731..750
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   909..1038
                     /label=AAV2 ITR
     promoter        1349..1626
                     /label=chicken beta-actin promoter
     primer_bind     1905..1927
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1989..2008
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2041..2050
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2047..2049,2050..2052,2053..2763)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2047..2049,2050..2052,2053..2763)
                     /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(2092..2113)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2353..2372)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2700..2721
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2899..3487
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2952..2972)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3370..3381)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3530..3651
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3541..3560)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3595..3614
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3813..3942
                     /label=AAV2 ITR
     rep_origin      complement(4179..4767)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4259..4278)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4938..5547,1..182,183..251))
                     /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 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
       61 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      121 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      181 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
      241 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
      301 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
      361 tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
      421 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
      481 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
      541 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
      601 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
      661 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
      721 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
      781 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
      841 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      901 tacgccagct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg
      961 cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact
     1021 ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc
     1081 catgctctag gaagagtacc attgacgtca ataatgacgt atgttcccat agtaacgcca
     1141 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     1201 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     1261 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     1321 tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc
     1381 cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt
     1441 gcagcgatgg gggcgggggg gggggggggg cgcgcgccag gcggggcggg gcggggcgrg
     1501 gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga
     1561 aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg
     1621 cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc
     1681 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct
     1741 tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg
     1801 cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggctg
     1861 tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg
     1921 tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca
     1981 gctcctgggc aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattggatcc
     2041 gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     2101 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     2161 acctacggca agctgaccct gaagttcatt tgcaccaccg gcaagctgcc cgtgccctgg
     2221 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     2281 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     2341 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     2401 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     2461 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     2521 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     2581 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     2641 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     2701 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     2761 aagggtggct ctggcggcag taccgacttc tatctcaagg gcgggtccgg aggctccaca
     2821 gatttttacc ttaaaggggg atccgggggt agcactgact tttatctcaa gtaagaattc
     2881 gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     2941 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     3001 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     3061 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     3121 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     3181 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     3241 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
     3301 tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc
     3361 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     3421 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     3481 tccccgcatc gataccgtcg acccgggcgg ccgcttcgag cagacatgat aagatacatt
     3541 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt
     3601 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     3661 aattgcattc attttatgtt tcaggttcag ggggagatgt gggaggtttt ttaaagcaag
     3721 taaaacctct acaaatgtgg taaaatcgat aaggatcttc ctagagcatg gctacgtaga
     3781 taagtagcat ggcgggttaa tcattaacta caaggaaccc ctagtgatgg agttggccac
     3841 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     3901 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcatta atgaatcggc
     3961 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4021 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4081 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4141 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4201 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4261 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4321 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4381 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4441 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4501 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4561 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4621 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4681 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4741 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4801 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4861 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4921 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4981 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5041 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5101 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5161 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5221 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5281 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5341 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5401 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5461 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5521 atgcggcgac cgagttgctc ttgcccg
//
