LOCUS       sequence-209568-        7144 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 7144)
  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 7144)
  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..7144
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        242..1419
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          473..1410
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1367..1387
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1450..1483)
                     /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(1534..1567)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1942..1953
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(2248..2295)
                     /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(2584..2640)
                     /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(2650..3375)
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="SKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRI
                     KVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVL
                     TATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDMA
                     LKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVAR
                     YCDLPSKLGHKLNGMDELYK"
     regulatory      3423..3432
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3437..3470
                     /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    3521..3554
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3574..4162
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3627..3647)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4045..4056)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4175..4399
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4407..4547
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4407..4536
                     /label=AAV2 ITR
     rep_origin      4622..5077
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4709..4728)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4919..4940
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5094..5113)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5213..5235
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5273..5291)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5359..5463
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5464..5532,5533..6324)
                     /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(5682..5701)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6495..7083
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6984..7003
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt
      181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
      241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
      481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
      541 tacttccact ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      601 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      661 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      721 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      781 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      841 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      901 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      961 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     1021 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     1081 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     1141 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     1201 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1261 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1321 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1381 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc
     1441 gactccggaa taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga
     1501 ttgtagctgc tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg
     1561 aagttatggc gcgccttagc tgtgggagtg ggggtgtgga tgcggatgag ggagagtatt
     1621 attctgtcca ccagtgaatg tgttgaaagt gtggagaggc tggattcctg gaatagtgtt
     1681 gggaatcatg gttatggtat tgggtgtcat caggggaatg tcgtctgggc ttcggcgcat
     1741 tgccagggta tagtccggag ggcatgctgt cctcagcacc acctcatggg gatggatgga
     1801 ttcacactcg tggtccaggt ctgtgtgttt catctgaaga ctcatgatct cctcctcagg
     1861 tgcgtgcgta agatcattgg ttgtagttcg ttgcggacta catctcctat gtacgtcgtg
     1921 gcgccttttg tccttcttgt aatagagggc cgcgaaagcc agaatgttca ggaaaagcag
     1981 ggaagctcct acagctatgg tcacggacag ctctgtgctg tagtcccttt ggtctacgct
     2041 aaatggacta ggctgttgtt tggggtcgtc ttgcttagcg gttggaaatg cgctggtcac
     2101 aggggttgag tttttccgag ttggtcgaag ggtgatgtca gtgcttggga ccttggtggt
     2161 tgtacttgtg tattggctga tgtcattgag gttatgcaag tggggcacca gctcgagggt
     2221 accgcttccg cctccggagc ctcctcctgt gattccagca gcgttcacgt actcgtgcag
     2281 caccatgtgg tctcttccgg agcctcctcc gctgcctcca ccggtgctgt tgacaggtgt
     2341 gatgtagttg ctagggaccc agccctggcc gttcttggtc tgggcctcgc accactcgcc
     2401 gttgtggttg tagcccagga ctctcagctt ctcgcccttg gtgatgctca gggtgttgtc
     2461 gccagaggcg acgaagtcgt acagggcgac gaacaggtta gggtcgttgc ctccgaccgg
     2521 tgcgtcgccg gtgctgccgg gcacccagag cagcagcacc catagcagga gggtgtcggt
     2581 ctcaggtcca gggttctcct ccacgtctcc agcctgcttc agcaggctga agttagtagc
     2641 tccgcttccc ttgtacagct cgtccatgcc attaagtttg tgccccagtt tgctagggag
     2701 gtcgcagtat ctggccacag ccacctcgtg ctgctcgacg taggtctctt tgtcggcctc
     2761 cttgattctt tccagtctgt ggtccacata gtagacgccg ggcatcttga ggttcttagc
     2821 gggtttcttg gatctgtatg tggtcttgaa gttgcagatc aggtggcccc cgcccacgag
     2881 cttcagggcc atgtcggttc tgccttccag gccgccgtca gcggggtaca gcatctcggt
     2941 gttggcctcc cagccgagtg ttttcttctg catcacaggg ccgttggatg ggaagttcac
     3001 ccctctgatc ttgacgttgt agatgaggca gccgtcctgg aggctggtgt cctgggtagc
     3061 ggtcagcacg cccccgtctt cgtatgtggt gactctctcc catgtgaagc cctcagggaa
     3121 ggactgctta aagaagtcgg ggatgccctg ggtgtggttg atgaaggttc tgctgccgta
     3181 catgaagctg gtagccagga tgtcgaaggc gaaggggaga gggccgccct cgaccacctt
     3241 gattctcatg gtctgggtgc cctcgtaggg cttgccttcg ccctcggatg tgcacttgaa
     3301 gtggtggttg ttcacggtgc cctccatgta cagcttcatg tgcatgttct ccttaatcag
     3361 ctcttcgccc ttagagcctc ctgtcagctt ggtagcctcc ttgtccttac actgcacaca
     3421 gcccatggtg gctagaataa cttcgtataa agtatcctat acgaagttat ttgccttaac
     3481 ccagaaatta tcactgttat tctttagaat ggtgcaaaga ataacttcgt ataatgtatg
     3541 ctatacgaag ttatgaattc ctgcagcccc gataatcaac ctctggatta caaaatttgt
     3601 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     3661 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     3721 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     3781 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     3841 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     3901 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     3961 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc
     4021 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     4081 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
     4141 tccctttggg ccgcctcccc gcatcggggg gatcctgtgc cttctagttg ccagccatct
     4201 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     4261 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     4321 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     4381 gatgcggtgg gctctatggc ggccgcagga acccctagtg atggagttgg ccactccctc
     4441 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     4501 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     4561 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     4621 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     4681 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     4741 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     4801 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     4861 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     4921 ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta
     4981 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     5041 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     5101 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     5161 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     5221 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     5281 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     5341 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     5401 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     5461 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     5521 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     5581 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     5641 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     5701 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     5761 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     5821 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     5881 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     5941 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     6001 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     6061 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     6121 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     6181 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     6241 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     6301 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     6361 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     6421 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     6481 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     6541 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     6601 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     6661 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     6721 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     6781 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     6841 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     6901 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     6961 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     7021 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     7081 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     7141 atgt
//
