LOCUS       sequence-212650-        7134 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An AAV vector that expresses double floxed myristoylated TagRFP-T 
            and post-eGRASP linked by self-cleaving P2A peptide under the Ef1a 
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7134)
  AUTHORS   Choi JH, Sim SE, Kim JI, Choi DI, Oh J, Ye S, Lee J, Kim T, Ko HG, 
            Lim CS, Kaang BK
  TITLE     Interregional synaptic maps among engram cells underlie memory 
            formation.
  JOURNAL   Science. 2018 Apr 27;360(6387):430-435. doi: 
            10.1126/science.aas9204.
  PUBMED    29700265
REFERENCE   2  (bases 1 to 7134)
  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: "10.1126/science"; 
            journalName: "Science"; date: "2018-04-27- 27"; volume: "360"; 
            issue: "6387"; pages: "430-435"
COMMENT     Sequence Label: pAAV-EWB-DIO-myrTagRFP-T-P2A-post-eGRASP
FEATURES             Location/Qualifiers
     source          1..7134
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1440..1473)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1524..1557)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1932..1943
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(2238..2285)
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     CDS             complement(2574..2630)
                     /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             complement(2640..3365)
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="SKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRI
                     KVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVL
                     TATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDMA
                     LKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVAR
                     YCDLPSKLGHKLNGMDELYK"
     regulatory      3413..3422
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3427..3460
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3511..3544
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3564..4152
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3617..3637)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4035..4046)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4165..4389
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4397..4537
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4397..4526
                     /label=AAV2 ITR
     rep_origin      4612..5067
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4699..4718)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4909..4930
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5084..5103)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5203..5225
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5263..5281)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5349..5453
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5454..5522,5523..6314)
                     /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(5672..5691)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6485..7073
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6974..6993
                     /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 gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggaa
     1441 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
     1501 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
     1561 gcgccttagc tgtgggagtg ggggtgtgga tgcggatgag ggagagtatt attctgtcca
     1621 ccagtgaatg tgttgaaagt gtggagaggc tggattcctg gaatagtgtt gggaatcatg
     1681 gttatggtat tgggtgtcat caggggaatg tcgtctgggc ttcggcgcat tgccagggta
     1741 tagtccggag ggcatgctgt cctcagcacc acctcatggg gatggatgga ttcacactcg
     1801 tggtccaggt ctgtgtgttt catctgaaga ctcatgatct cctcctcagg tgcgtgcgta
     1861 agatcattgg ttgtagttcg ttgcggacta catctcctat gtacgtcgtg gcgccttttg
     1921 tccttcttgt aatagagggc cgcgaaagcc agaatgttca ggaaaagcag ggaagctcct
     1981 acagctatgg tcacggacag ctctgtgctg tagtcccttt ggtctacgct aaatggacta
     2041 ggctgttgtt tggggtcgtc ttgcttagcg gttggaaatg cgctggtcac aggggttgag
     2101 tttttccgag ttggtcgaag ggtgatgtca gtgcttggga ccttggtggt tgtacttgtg
     2161 tattggctga tgtcattgag gttatgcaag tggggcacca gctcgagggt accgcttccg
     2221 cctccggagc ctcctcctgt gattccagca gcgttcacgt actcgtgcag caccatgtgg
     2281 tctcttccgg agcctcctcc gctgcctcca ccggtgctgt tgacaggtgt gatgtagttg
     2341 ctagggaccc agccctggcc gttcttggtc tgggcctcgc accactcgcc gttgtggttg
     2401 tagcccagga ctctcagctt ctcgcccttg gtgatgctca gggtgttgtc gccagaggcg
     2461 acgaagtcgt acagggcgac gaacaggtta gggtcgttgc ctccgaccgg tgcgtcgccg
     2521 gtgctgccgg gcacccagag cagcagcacc catagcagga gggtgtcggt ctcaggtcca
     2581 gggttctcct ccacgtctcc agcctgcttc agcaggctga agttagtagc tccgcttccc
     2641 ttgtacagct cgtccatgcc attaagtttg tgccccagtt tgctagggag gtcgcagtat
     2701 ctggccacag ccacctcgtg ctgctcgacg taggtctctt tgtcggcctc cttgattctt
     2761 tccagtctgt ggtccacata gtagacgccg ggcatcttga ggttcttagc gggtttcttg
     2821 gatctgtatg tggtcttgaa gttgcagatc aggtggcccc cgcccacgag cttcagggcc
     2881 atgtcggttc tgccttccag gccgccgtca gcggggtaca gcatctcggt gttggcctcc
     2941 cagccgagtg ttttcttctg catcacaggg ccgttggatg ggaagttcac ccctctgatc
     3001 ttgacgttgt agatgaggca gccgtcctgg aggctggtgt cctgggtagc ggtcagcacg
     3061 cccccgtctt cgtatgtggt gactctctcc catgtgaagc cctcagggaa ggactgctta
     3121 aagaagtcgg ggatgccctg ggtgtggttg atgaaggttc tgctgccgta catgaagctg
     3181 gtagccagga tgtcgaaggc gaaggggaga gggccgccct cgaccacctt gattctcatg
     3241 gtctgggtgc cctcgtaggg cttgccttcg ccctcggatg tgcacttgaa gtggtggttg
     3301 ttcacggtgc cctccatgta cagcttcatg tgcatgttct ccttaatcag ctcttcgccc
     3361 ttagagcctc ctgtcagctt ggtagcctcc ttgtccttac actgcacaca gcccatggtg
     3421 gctagaataa cttcgtataa agtatcctat acgaagttat ttgccttaac ccagaaatta
     3481 tcactgttat tctttagaat ggtgcaaaga ataacttcgt ataatgtatg ctatacgaag
     3541 ttatgaattc ctgcagcccc gataatcaac ctctggatta caaaatttgt gaaagattga
     3601 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     3661 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     3721 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     3781 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     3841 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     3901 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     3961 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     4021 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     4081 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     4141 ccgcctcccc gcatcggggg gatcctgtgc cttctagttg ccagccatct gttgtttgcc
     4201 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     4261 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     4321 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     4381 gctctatggc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     4441 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     4501 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     4561 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     4621 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     4681 ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     4741 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     4801 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     4861 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     4921 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt
     4981 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     5041 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     5101 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     5161 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     5221 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     5281 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     5341 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     5401 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     5461 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     5521 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     5581 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     5641 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     5701 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     5761 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     5821 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     5881 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     5941 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     6001 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     6061 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6121 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     6181 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     6241 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     6301 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     6361 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     6421 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     6481 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6541 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6601 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     6661 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6721 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6781 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6841 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6901 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     6961 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     7021 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     7081 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
