LOCUS       sequence-485807-        5573 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV transfer plasmid expressing eGFP-NWS under a CAG promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5573)
  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 5573)
  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-NWS
FEATURES             Location/Qualifiers
     source          1..5573
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     132..151
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(370..474)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(500..955)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(637..658)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     849..868
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   1027..1156
                     /label=AAV2 ITR
     primer_bind     2022..2044
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2106..2125
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2158..2167
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2164..2166,2167..2169,2170..2880)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2164..2166,2167..2169,2170..2880)
                     /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(2209..2230)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2470..2489)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2817..2838
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2902..2925
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2947..2970
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2992..3015
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     misc_feature    3043..3631
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3096..3116)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3514..3525)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3674..3795
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3685..3704)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3739..3758
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3957..4086
                     /label=AAV2 ITR
     rep_origin      complement(4323..4911)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4403..4422)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5082..5573,1..300,301..369))
                     /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 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
       61 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
      121 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
      181 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
      241 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
      301 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
      361 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
      421 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
      481 tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
      541 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
      601 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
      661 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
      721 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
      781 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
      841 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
      901 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat
      961 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     1021 cgccagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg
     1081 acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc
     1141 atcactaggg gttccttgta gttaatgatt aacccgccat gctacttatc tacgtagcca
     1201 tgctctagga agagtaccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     1261 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     1321 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     1381 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     1441 cgtattagtc atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc
     1501 catctccccc ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc
     1561 agcgatgggg gcgggggggg gggggggggc ggcccggggg gggggggggg gggggggggg
     1621 gggggggggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag
     1681 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
     1741 gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc
     1801 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct
     1861 cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt
     1921 gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggctgtcc
     1981 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     2041 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     2101 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tggatccgcc
     2161 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     2221 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     2281 tacggcaagc tgaccctgaa gttcatttgc accaccggca agctgcccgt gccctggccc
     2341 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     2401 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     2461 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     2521 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     2581 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     2641 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     2701 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     2761 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     2821 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     2881 gggggatctg gcggttctaa ttggtcacac ccccagttcg agaaaggtgg ttccggcggt
     2941 agcaactgga gccatcctca attcgaaaaa gggggaagcg ggggcagcaa ttggagccac
     3001 ccacaatttg aaaaataaga attcgatatc aagcttatcg ataatcaacc tctggattac
     3061 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     3121 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     3181 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     3241 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     3301 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     3361 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     3421 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg
     3481 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     3541 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     3601 agtcggatct ccctttgggc cgcctccccg catcgatacc gtcgacccgg gcggccgctt
     3661 cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga
     3721 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     3781 tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag
     3841 atgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtaaaat cgataaggat
     3901 cttcctagag catggctacg tagataagta gcatggcggg ttaatcatta actacaagga
     3961 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     4021 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     4081 gcgcagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4141 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4201 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4261 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4321 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4381 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4441 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4501 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4561 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4621 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4681 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4741 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4801 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4861 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4921 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4981 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     5041 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     5101 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     5161 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     5221 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     5281 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     5341 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     5401 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     5461 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     5521 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggc
//
