LOCUS       sequence-209571-        7399 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An AAV vector that expresses double floxed myristoylated iRFP670 
            with V5 tag 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 7399)
  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 7399)
  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-myriRFP670V5-P2A-post-eGRASP
FEATURES             Location/Qualifiers
     source          1..7399
                     /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..2691)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(2698..3627)
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 670 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP670
                     /note="derived from Rhodopseudomonas palustris RpBphP6"
                     /translation="ARKVDLTSCDREPIHIPGSIQPCGCLLACDAQAVRITRITENAGA
                     FFGRETPRVGELLADYFGETEAHALRNALAQSSDPKRPALIFGWRDGLTGRTFDISLHR
                     HDGTSIIEFEPAAAEQADNPLRLTRQIIARTKELKSLEEMAARVPRYLQAMLGYHRVML
                     YRFADDGSGMVIGEAKRSDLESFLGQHFPASLVPQQARLLYLKNAIRVVSDSRGISSRI
                     VPEHDASGAALDLSFAHLRSISPCHLEFLRNMGVSASMSLSIIIDGTLWGLIICHHYEP
                     RAVPMAQRVAAEMFADFLSLHFTAAHHQR"
     regulatory      3678..3687
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3692..3725
                     /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    3776..3809
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3829..4417
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3882..3902)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4300..4311)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4430..4654
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4662..4802
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4662..4791
                     /label=AAV2 ITR
     rep_origin      4877..5332
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4964..4983)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5174..5195
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5349..5368)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5468..5490
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5528..5546)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5614..5718
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5719..5787,5788..6579)
                     /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(5937..5956)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6750..7338
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7239..7258
                     /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 tccgcttcct gtagagtcca gtcccagcag aggattaggg ataggcttgc cggatccgcg
     2701 ttggtggtgg gcggcggtga agtgcagcga taagaagtcg gcgaacattt cggccgcgac
     2761 gcgctgcgcc atcggcacgg cacgcggctc gtaatgatga cagatgatca atccccatag
     2821 cgtgccgtca atgatgatcg acagcgacat cgaggcgctg acgcccatgt tccgcagaaa
     2881 ttcgagatgg cagggcgaga tgctgcgcag gtgcgcgaac gacagatcga gcgcggcgcc
     2941 ggaggcgtcg tgctcgggca cgatccggct gctgatgccg cgcgaatccg agaccacgcg
     3001 gatcgcgttc ttcaagtaca gtagccgcgc ctgctgcggg accagcgacg ccggaaagtg
     3061 ctgaccgaga aagctctcga ggtcgctgcg cttcgcctcg ccgatcacca tcccggagcc
     3121 gtcgtccgcg aagcggtaca acatcacgcg gtgatagccg agcatcgcct gcagatagcg
     3181 cggcacccgt gcggccatct cttcgagcga cttcagttct ttggtgcgcg cgatgatctg
     3241 ccgcgtcagc cgcagcggat tgtcggcctg ttcggccgcc gcaggctcga actcgatgat
     3301 cgatgtaccg tcatggcgat gcagtgagat gtcgaaggtg cggccggtca ggccgtcgcg
     3361 ccaaccgaag atcagcgccg gtcgctttgg atcggaggac tgcgccagtg cgttgcgcag
     3421 cgcatgggct tcggtctcgc cgaagtaatc ggcgagtagc tcaccgaccc gcggagtttc
     3481 gcgtccaaag aacgcgccgg cattttccgt aatgcgcgtg atccgcaccg cctgcgcgtc
     3541 gcaggctagc aggcagccgc acggctgaat gctgccgggg atgtggatcg gctcgcgatc
     3601 gcaggaggtg agatcgacct tacgcgcaga gcctcctgtc agcttggtag cctccttgtc
     3661 cttacactgc acacagccca tggtggctag aataacttcg tataaagtat cctatacgaa
     3721 gttatttgcc ttaacccaga aattatcact gttattcttt agaatggtgc aaagaataac
     3781 ttcgtataat gtatgctata cgaagttatg aattcctgca gccccgataa tcaacctctg
     3841 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     3901 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     3961 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     4021 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     4081 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     4141 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     4201 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc
     4261 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     4321 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     4381 cagacgagtc ggatctccct ttgggccgcc tccccgcatc ggggggatcc tgtgccttct
     4441 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     4501 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     4561 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     4621 agcaggcatg ctggggatgc ggtgggctct atggcggccg caggaacccc tagtgatgga
     4681 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     4741 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     4801 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     4861 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4921 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     4981 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     5041 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     5101 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     5161 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc
     5221 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     5281 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     5341 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     5401 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     5461 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     5521 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     5581 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     5641 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     5701 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     5761 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     5821 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     5881 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     5941 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     6001 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     6061 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     6121 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     6181 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     6241 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     6301 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6361 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     6421 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     6481 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     6541 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     6601 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6661 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6721 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6781 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6841 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     6901 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6961 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     7021 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     7081 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     7141 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     7201 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7261 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     7321 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7381 tggccttttg ctcacatgt
//
