LOCUS       sequence-425139-        5681 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of SYFP2 (yellow fluorescent protein) plus separate 
            nuclear FLAG epitope in striatal cholinergic neurons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5681)
  AUTHORS   Hunker AC, Wirthlin ME, Gill G, Johansen NJ, Hooper M, Omstead V, 
            Vargas S, Lerma MN, Taskin N, Weed N, Laird WD, Bishaw YM, Bendrick 
            JL, Gore BB, Ben-Simon Y, Opitz-Araya X, Martinez RA, Way SW, 
            Thyagarajan B, Otto S, Sanchez REA, Alexander JR, Amaya A, Amster A,
            Arbuckle J, Ayala A, Baker PM, Barcelli T, Barta S, Bertagnolli D, 
            Bielstein C, Bishwakarma P, Bowlus J, Boyer G, Brouner K, Casian B, 
            Casper T, Chakka AB, Chakrabarty R, Chong P, Clark M, Colbert K, 
            Daniel S, Dawe T, Departee M, DiValentin P, Donadio NP, Dotson NI, 
            Dwivedi D, Egdorf T, Fliss T, Gary A, Goldy J, Grasso C, Groce EL, 
            Gudsnuk K, Han W, Haradon Z, Hastings S, Helback O, Ho WV, Huang C, 
            Johnson T, Jones DL, Juneau Z, Kenney J, Leibly M, Li S, Liang E, 
            Loeffler H, Lusk NA, Madigan Z, Malloy J, Malone J, McCue R, Melchor
            J, Mich JK, Moosman S, Morin E, Naidoo R, Newman D, Ngo K, Nguyen K,
            Oster AL, Ouellette B, Oyama AA, Pena N, Pham T, Phillips E, Pom C, 
            Potekhina L, Ransford S, Ray PL, Reding M, Rette DF, Reynoldson C, 
            Rimorin C, Sigler AR, Rocha DB, Ronellenfitch K, Ruiz A, Sawyer L, 
            Sevigny JP, Shapovalova NV, Shepard N, Shulga L, Soliman S, Staats 
            B, Taormina MJ, Tieu M, Wang Y, Wilkes J, Wood T, Zhou T, Williford 
            A, Dee N, Mollenkopf T, Ng L, Esposito L, Kalmbach BE, Yao S, Ariza 
            J, Collman F, Mufti S, Smith K, Waters J, Ersing I, Patrick M, Zeng 
            H, Lein ES, Kojima Y, Horwitz G, Owen SF, Levi BP, Daigle TL, Tasic 
            B, Bakken TE, Ting JT
  TITLE     Enhancer AAV toolbox for accessing and perturbing striatal cell 
            types and circuits.
  JOURNAL   Neuron. 2025 May 21;113(10):1507-1524.e17. doi: 
            10.1016/j.neuron.2025.04.035.
  PUBMED    40403704
REFERENCE   2  (bases 1 to 5681)
  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"; doi: 
            "10.1016/j.neuron.2025.04"; journalName: "Neuron"; date: 
            "2025-05-21- 21"; volume: "113"; issue: "10"; pages: "1507-1524.e17"
COMMENT     Sequence Label: AiP14496:
            pAAV-AiE0873m_3xC2-minBG-SYFP2-P2A-3...
FEATURES             Location/Qualifiers
     source          1..5681
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      1147..1156
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1153..1155,1156..1158,1159..1869)
                     /codon_start=1
                     /product="monomeric optimized variant of EYFP, identical to
                     mVenus but with L68V (Kremers et al., 2006)"
                     /label=SYFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1459..1478)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1806..1827
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1879..1935
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1948..2013
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2044..2421
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAITKYTSAK"
     primer_bind     complement(2508..2528)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    2715..2826
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2944..3084
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2944..3073
                     /label=AAV2 ITR
     rep_origin      3159..3614
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3246..3265)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3456..3477
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3631..3650)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3750..3772
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3810..3828)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3896..4000
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4001..4069,4070..4861)
                     /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"
     primer_bind     complement(4219..4238)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5032..5620
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5521..5540
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtcatgga tttctccaaa gctttgtagg gcagaactct
      181 gaatgtttat ttctctgatg aatccttaat gaaagtggcc agccatcctg gggagaacaa
      241 gccttagttc ccactgagaa ggatagcaag cagcataaga gtagctgatg tcaaaggcta
      301 ttaatgcaaa tgaaaattat aattcctcat caagatctgc tcccagacag gctaatcatt
      361 gtgccacctg tttacagaaa cacactgcta atcagtcagt aaaatctttt aagtttctgt
      421 gcttctctgt gtacgatttt tagcctccat ggatttctcc aaagctttgt agggcagaac
      481 tctgaatgtt tatttctctg atgaatcctt aatgaaagtg gccagccatc ctggggagaa
      541 caagccttag ttcccactga gaaggatagc aagcagcata agagtagctg atgtcaaagg
      601 ctattaatgc aaatgaaaat tataattcct catcaagatc tgctcccaga caggctaatc
      661 attgtgccac ctgtttacag aaacacactg ctaatcagtc agtaaaatct tttaagtttc
      721 tgtgcttctc tgtgtacgat ttttagcctc catggatttc tccaaagctt tgtagggcag
      781 aactctgaat gtttatttct ctgatgaatc cttaatgaaa gtggccagcc atcctgggga
      841 gaacaagcct tagttcccac tgagaaggat agcaagcagc ataagagtag ctgatgtcaa
      901 aggctattaa tgcaaatgaa aattataatt cctcatcaag atctgctccc agacaggcta
      961 atcattgtgc cacctgttta cagaaacaca ctgctaatca gtcagtaaaa tcttttaagt
     1021 ttctgtgctt ctctgtgtac gatttttagc ctcgagctcg ggctgggcat aaaagtcagg
     1081 gcagagccat ctattgctta catttgcttc tgggatccag atctttcgaa gctagcgcta
     1141 ccggtcgcca ccatggtctc caagggcgag gagctgttca ccggggtggt gcccatcctg
     1201 gtcgagctgg acggcgatgt caacggccac aagttcagcg tgtccggcga gggcgagggc
     1261 gatgccacct acggcaagct gaccctgaag ctgatctgca ccaccggcaa gctgcccgtg
     1321 ccctggccca ccctcgtgac caccctgggc tacggcgtgc agtgcttcgc ccgctacccc
     1381 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     1441 cgcaccatct tcttcaaaga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     1501 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1561 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat caccgccgac
     1621 aagcagaaga acggcatcaa ggccaacttc aagatccgcc acaacatcga ggacggcggc
     1681 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1741 cccgacaacc actacctgag ctaccagtcc aagctgagca aagaccccaa cgagaagcgc
     1801 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1861 ctgtacaagg gctctggtgc taccaacttc tcactgttga aacaggcagg ggatgtagag
     1921 gagaatccag ggcctggtgc tagtggagac tacaaagacc atgacggaga ttataaagat
     1981 catgacatcg attacaagga tgacgatgac aagtccggac tcagatctgg aggctccgga
     2041 ggcccagagc cagcgaagtc tgctcccgcc ccgaaaaagg gctccaagaa ggcggtgact
     2101 aaggcgcaga agaaaggcgg caagaagcgc aagcgcagcc gcaaggagag ctattccatc
     2161 tatgtgtata aggttctgaa gcaggtccac cctgacaccg gcatttcgtc caaggccatg
     2221 ggcatcatga actcgtttgt gaacgacatt ttcgagcgca tcgcaggtga ggcttcccgc
     2281 ctggcgcatt acaacaagcg ctcgaccatc acctccaggg agatccagac ggccgtgcgc
     2341 ctgctgctgc ctggggagtt ggccaagcac gccgtgtccg agggtactaa ggccatcacc
     2401 aagtacacca gcgctaagta atgaggcgcg ccgcggccgc gaattcgata tcataatcaa
     2461 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     2521 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     2581 ttcattttct cctccttgta taaatcctgg ttagttcttg ccacggcgga actcatcgcc
     2641 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggct
     2701 cgagagatct tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     2761 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     2821 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     2881 caagggggag gattgggaag acaatagcag gcatgagatc tcacgtgcgg accgagcggc
     2941 cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     3001 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     3061 agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt
     3121 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     3181 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc
     3241 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3301 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3361 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3421 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3481 aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt
     3541 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     3601 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     3661 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     3721 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     3781 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     3841 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     3901 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3961 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     4021 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     4081 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     4141 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     4201 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     4261 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     4321 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     4381 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     4441 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     4501 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     4561 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     4621 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     4681 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     4741 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     4801 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     4861 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     4921 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     4981 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     5041 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     5101 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     5161 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     5221 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     5281 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     5341 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     5401 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     5461 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     5521 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     5581 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     5641 tttacggttc ctggcctttt gctggccttt tgctcacatg t
//
