LOCUS       sequence-486019-        5663 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV transfer plasmid expressing eGFP-SunTag under a CAG promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5663)
  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 5663)
  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-SunTag
FEATURES             Location/Qualifiers
     source          1..5663
                     /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"
     CDS             2789..2845
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             2864..2920
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             2939..2995
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     misc_feature    3023..3611
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3076..3096)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3494..3505)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3654..3775
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3665..3684)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3719..3738
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3937..4066
                     /label=AAV2 ITR
     rep_origin      complement(4303..4891)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4383..4402)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5062..5663,1..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 gggggggsgc gcgccrgggg gggsgggggg
     1501 gggsgggggg sggggsgggg sgrggcggrg aggtgcggcg 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 ggtggaagtg gcggcagcga agaactcctg tcaaagaatt atcacctgga
     2821 aaatgaggtg gctagactta agaaaggggg ttccggtggc agtgaagagc tgctctctaa
     2881 gaactatcac ttggagaacg aggttgctcg attgaaaaaa ggagggagcg gcggttcaga
     2941 agaattgctc tccaaaaact accatctcga gaatgaagtt gcccgactta aaaagtaaga
     3001 attcgatatc aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac
     3061 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     3121 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     3181 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     3241 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     3301 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     3361 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc
     3421 atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
     3481 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     3541 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     3601 cgcctccccg catcgatacc gtcgacccgg gcggccgctt cgagcagaca tgataagata
     3661 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     3721 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     3781 caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag
     3841 caagtaaaac ctctacaaat gtggtaaaat cgataaggat cttcctagag catggctacg
     3901 tagataagta gcatggcggg ttaatcatta actacaagga acccctagtg atggagttgg
     3961 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     4021 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc attaatgaat
     4081 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4141 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4201 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4261 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4321 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4381 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4441 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4501 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4561 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4621 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4681 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4741 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4801 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4861 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4921 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4981 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5041 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5101 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5161 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5221 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5281 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5341 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5401 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5461 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5521 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5581 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5641 gtgtatgcgg cgaccgagtt gct
//
