LOCUS       sequence-485800-        5609 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV transfer plasmid expressing eGFP-ETAG under a CAG promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5609)
  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 5609)
  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-ETAG
FEATURES             Location/Qualifiers
     source          1..5609
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     22..41
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(260..364)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(390..845)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(527..548)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     739..758
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   917..1046
                     /label=AAV2 ITR
     primer_bind     1912..1934
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1996..2015
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2048..2057
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2054..2056,2057..2059,2060..2770)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2054..2056,2057..2059,2060..2770)
                     /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(2099..2120)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2360..2379)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2707..2728
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2969..3557
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3022..3042)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3440..3451)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3600..3721
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3611..3630)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3665..3684
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3883..4012
                     /label=AAV2 ITR
     rep_origin      complement(4249..4837)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4329..4348)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5008..5609,1..190,191..259))
                     /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 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
       61 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
      121 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
      181 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
      241 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
      301 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
      361 cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt
      421 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
      481 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
      541 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
      601 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
      661 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
      721 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
      781 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
      841 cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
      901 ttcgctatta cgccagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg
      961 gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt
     1021 ggccaactcc atcactaggg gttccttgta gttaatgatt aacccgccat gctacttatc
     1081 tacgtagcca tgctctagga agagtaccat tgacgtcaat aatgacgtat gttcccatag
     1141 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     1201 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     1261 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     1321 agtacatcta cgtattagtc atcgctatta ccatggtcga ggtgagcccc acgttctgct
     1381 tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt attttttaat
     1441 tattttgtgc agcgatgggg gcgggggggg ggggggggcg cgccrggggg gggggggggg
     1501 gggggggggg gggggggggg gggggggggg gggggsggcg gcagccaatc agagcggcgc
     1561 gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag
     1621 cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc
     1681 ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga
     1741 cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct
     1801 gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc
     1861 ggggctgtcc gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct
     1921 tctggcgtgt gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt
     1981 tcctacagct cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat
     2041 tggatccgcc accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     2101 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     2161 cgatgccacc tacggcaagc tgaccctgaa gttcatttgc accaccggca agctgcccgt
     2221 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     2281 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     2341 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     2401 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     2461 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     2521 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     2581 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     2641 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     2701 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     2761 gctgtacaag ggaggaagcg ggggatccgg ggctcctgtc ccatatcctg atccccttga
     2821 gccccgggga ggtagtggag ggtctggagc tcctgttcct taccctgatc cattggaacc
     2881 taggggaggc agtggtggaa gtggcgcccc agtcccctac cccgatcctc ttgaaccccg
     2941 ataagaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaagg
     3001 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     3061 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     3121 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     3181 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     3241 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     3301 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     3361 gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     3421 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     3481 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     3541 ttgggccgcc tccccgcatc gataccgtcg acccgggcgg ccgcttcgag cagacatgat
     3601 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     3661 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     3721 taacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt gggaggtttt
     3781 ttaaagcaag taaaacctct acaaatgtgg taaaatcgat aaggatcttc ctagagcatg
     3841 gctacgtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc ctagtgatgg
     3901 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     3961 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcatta
     4021 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4081 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4141 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4201 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4261 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4321 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4381 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4441 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4501 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4561 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4621 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4681 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4741 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4801 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4861 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4921 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4981 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5041 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5101 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5161 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5221 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5281 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5341 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5401 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5461 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5521 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5581 agaatagtgt atgcggcgac cgagttgct
//
