LOCUS       sequence-486117-        5608 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV transfer plasmid expressing eGFP-ALFA under a CAG promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5608)
  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 5608)
  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-ALFA
FEATURES             Location/Qualifiers
     source          1..5608
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(96..115)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      909..1497
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1398..1417
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1734..1863
                     /label=AAV2 ITR
     polyA_signal    2025..2146
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2062..2081)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2116..2135
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    2189..2777
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             2295..2306
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     2704..2724
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2805..2843)
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             complement(2862..2900)
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             complement(2919..2957)
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             complement(join(2976..3686,3687..3689,3690..3692))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(2976..3686,3687..3689,3690..3692))
                     /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(3018..3039)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3367..3386
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3626..3647
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3689..3698
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3731..3750)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(3812..3834)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(4113..4388)
                     /label=chicken beta-actin promoter
     repeat_region   4699..4828
                     /label=AAV2 ITR
     rep_origin      4900..5355
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4987..5006)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5197..5218
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5381..5485
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5486..5554,5555..5608,1..738)
                     /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 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
       61 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
      121 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
      181 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
      241 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
      301 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
      361 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
      421 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
      481 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
      541 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
      601 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
      661 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
      721 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
      781 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
      841 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
      901 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
      961 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     1021 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     1081 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     1141 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     1201 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     1261 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     1321 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     1381 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     1441 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     1501 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     1561 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     1621 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     1681 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctgcgcg
     1741 ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc
     1801 cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt
     1861 ccttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc tctaggaaga
     1921 tccttatcga ttttaccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacatc
     1981 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     2041 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     2101 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgctcga
     2161 agcggccgcc cgggtcgacg gtatcgatgc ggggaggcgg cccaaaggga gatccgactc
     2221 gtctgagggc gaaggcgaag acgcggaaga ggccgcagag ccggcagcag gccgcgggaa
     2281 ggaaggtccg ctggattgag ggccgaaggg acgtagcaga aggacgtccc gcgcagaatc
     2341 caggtggcaa cacaggcgag cagccaagga aaggacgatg atttccccga caacaccacg
     2401 gaattgtcag tgcccaacag ccgagcccct gtccagcagc gggcaaggca ggcggcgatg
     2461 agttccgccg tggcaatagg gagggggaaa gcgaaagtcc cggaaaggag ctgacaggtg
     2521 gtggcaatgc cccaaccagt gggggttgcg tcagcaaaca cagtgcacac cacgccacgt
     2581 tgcctgacaa cgggccacaa ctcctcataa agagacagca accaggattt atacaaggag
     2641 gagaaaatga aagccatacg ggaagcaata gcatgataca aaggcattaa agcagcgtat
     2701 ccacatagcg taaaaggagc aacatagtta agaataccag tcaatctttc acaaattttg
     2761 taatccagag gttgattatc gataagcttg atatcgaatt cttattctgt aagtcttctc
     2821 ctaagctcct cctcgagtct agaagaacct cctgagcctc cttctgtcaa tcggcgtcta
     2881 agttcctcct ccaggcggga agatccaccg ctaccacctt cagtcaacct gcgtcggagc
     2941 tcttcttcga gccgtgaaga tcctccgctt ccacccttgt acagctcgtc catgccgaga
     3001 gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt ctcgttgggg
     3061 tctttgctca gggcggactg ggtgctcagg tagtggttgt cgggcagcag cacggggccg
     3121 tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct gcacgctgcc gtcctcgatg
     3181 ttgtggcgga tcttgaagtt caccttgatg ccgttcttct gcttgtcggc catgatatag
     3241 acgttgtggc tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc gtcctccttg
     3301 aagtcgatgc ccttcagctc gatgcggttc accagggtgt cgccctcgaa cttcacctcg
     3361 gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct
     3421 tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta gcggctgaag
     3481 cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc agggcacggg cagcttgccg
     3541 gtggtgcaaa tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg
     3601 gacacgctga acttgtggcc gtttacgtcg ccgtccagct cgaccaggat gggcaccacc
     3661 ccggtgaaca gctcctcgcc cttgctcacc atggtggcgg atccaattct ttgccaaaat
     3721 gatgagacag cacaataacc agcacgttgc ccaggagctg taggaaaaag aagaaggcat
     3781 gaacatggtt agcagaggct ctagagccgc cggtcacacg ccagaagccg aaccccgccc
     3841 tgccccgtcc cccccgaagg cagccgtccc cccgcggaca gccccgagcc gctccccccg
     3901 cacaaagggc cctcccggag cccctcaagg ctttcacgca gccacagaaa agaaacaagc
     3961 cgtcattaaa ccaagcgcta attacagccc ggaggagaag ggccgtcccg cccgctcacc
     4021 tgtgggagta acgcggtcag tcagagccgg ggcgggcggc gcgaggcggc ggcggagcgg
     4081 ggcacggggc gaaggcagcg cgcagcgact cccgcccgcc gcgcgcttcg ctttttatag
     4141 ggccgccgcc gccgccgcct cgccataaaa ggaaactttc ggagcgcgcc gctctgattg
     4201 gctgccgccg cacctctccg cctcgccccg ccccgcccct cgccccgccc cgccccgcct
     4261 ggcgcgcgcc cccccccccc ccgcccccat cgctgcacaa aataattaaa aaataaataa
     4321 atacaaaatt gggggtgggg agggggggga gatggggaga gtgaagcaga acgtggggct
     4381 cacctcgacc atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc
     4441 ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat
     4501 agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa
     4561 atactccacc cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt
     4621 cattattgac gtcaatggta ctcttcctag agcatggcta cgtagataag tagcatggcg
     4681 ggttaatcat taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     4741 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     4801 cggcctcagt gagcgagcga gcgcgcagct ggcgtaatag cgaagaggcc cgcaccgatc
     4861 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat
     4921 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     4981 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     5041 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     5101 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     5161 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     5221 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     5281 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     5341 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5401 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5461 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5521 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5581 agatgctgaa gatcagttgg gtgcacga
//
