LOCUS       sequence-486020-        5601 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV transfer plasmid expressing eGFP-SPOT under a CAG promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5601)
  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 5601)
  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-SPOT
FEATURES             Location/Qualifiers
     source          1..5601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     10..29
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(248..352)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(378..833)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(515..536)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     727..746
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   905..1034
                     /label=AAV2 ITR
     primer_bind     1901..1923
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1985..2004
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2037..2046
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2043..2045,2046..2048,2049..2759)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2043..2045,2046..2048,2049..2759)
                     /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(2088..2109)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2349..2368)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2696..2717
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2778..2813
                     /codon_start=1
                     /product="engineered Spot-Tag(R) peptide that binds with 
                     high affinity to a nanobody"
                     /label=Spot-Tag
                     /translation="PDRVRAVSHWSS"
     CDS             2832..2867
                     /codon_start=1
                     /product="engineered Spot-Tag(R) peptide that binds with 
                     high affinity to a nanobody"
                     /label=Spot-Tag
                     /translation="PDRVRAVSHWSS"
     CDS             2886..2921
                     /codon_start=1
                     /product="engineered Spot-Tag(R) peptide that binds with 
                     high affinity to a nanobody"
                     /label=Spot-Tag
                     /translation="PDRVRAVSHWSS"
     misc_feature    2949..3537
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3002..3022)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3420..3431)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3580..3701
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3591..3610)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3645..3664
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3863..3992
                     /label=AAV2 ITR
     rep_origin      complement(4229..4817)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4309..4328)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4988..5601,1..178,179..247))
                     /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 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
       61 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
      121 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
      181 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
      241 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
      301 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
      361 cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
      421 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
      481 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
      541 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
      601 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
      661 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
      721 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
      781 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc
      841 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
      901 ccagctgcgc gctcgctcgc tcactgaggc cgcccgggca aagcccgggc gtcgggcgac
      961 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat
     1021 cactaggggt tccttgtagt taatgattaa cccgccatgc tacttatcta cgtagccatg
     1081 ctctaggaag agtaccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     1141 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     1201 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     1261 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     1321 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
     1381 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
     1441 cgatgggggc gggggggggg ggggggcgcg cgccgggggg gggggggggg ggsggggggs
     1501 ggggsggggs grggsggrga ggkgcggcgg cagccaatca gagcggcgcg ctccgaaagt
     1561 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
     1621 cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
     1681 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
     1741 ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg
     1801 aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggctgtccg
     1861 cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg
     1921 accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc
     1981 ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt ggatccgcca
     2041 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     2101 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     2161 acggcaagct gaccctgaag ttcatttgca ccaccggcaa gctgcccgtg ccctggccca
     2221 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     2281 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     2341 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     2401 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     2461 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     2521 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     2581 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     2641 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     2701 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg
     2761 gtggttccgg tggttctcca gacagagtaa gggccgtatc acactggagt agcggcggat
     2821 ctggtggttc acccgacagg gttagggcag tgtcacattg gtcaagcggg ggaagtgggg
     2881 gatctcctga ccgcgtacgc gcagtatccc attggtcatc ataagaattc gatatcaagc
     2941 ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     3001 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     3061 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     3121 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     3181 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     3241 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     3301 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt
     3361 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     3421 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     3481 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc
     3541 gataccgtcg acccgggcgg ccgcttcgag cagacatgat aagatacatt gatgagtttg
     3601 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     3661 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     3721 attttatgtt tcaggttcag ggggagatgt gggaggtttt ttaaagcaag taaaacctct
     3781 acaaatgtgg taaaatcgat aaggatcttc ctagagcatg gctacgtaga taagtagcat
     3841 ggcgggttaa tcattaacta caaggaaccc ctagtgatgg agttggccac tccctctctg
     3901 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     3961 cgggcggcct cagtgagcga gcgagcgcgc agctgcatta atgaatcggc caacgcgcgg
     4021 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4081 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4141 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4201 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4261 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4321 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4381 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4441 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4501 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4561 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4621 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4681 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4741 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4801 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4861 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4921 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4981 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5041 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5101 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5161 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5221 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5281 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5341 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5401 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5461 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5521 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5581 cgagttgctc ttgcccggcg t
//
