LOCUS       Exported                7389 bp ds-DNA     circular SYN 12-MAY-2021
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    pAAV-EWB-DIO-myriRFP670V5-P2A-post-eGRASP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7389)
  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 7389)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7389
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..1179
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          232..1170
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1127..1147
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1210..1243
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    1294..1327
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             1702..1713
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(2008..2055)
                     /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(2344..2400)
                     /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(2410..2451)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(2458..3387)
                     /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      3438..3447
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(3452..3485)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    complement(3536..3569)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    3589..4177
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3642..3662)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4060..4071)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4190..4414
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4422..4562
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4422..4551
                     /label=AAV2 ITR
     rep_origin      4637..5092
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4724..4743)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4934..4955
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5109..5128)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5228..5250
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5288..5306)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5374..5478
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5479..6339
                     /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(5697..5716)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6510..7098
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6999..7018
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7160..7289
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
       61 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
      121 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
      181 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
      241 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
      301 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      361 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      421 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      481 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      541 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      601 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      661 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      721 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
      781 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
      841 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
      901 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
      961 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1021 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1081 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1141 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc
     1201 gactccggaa taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga
     1261 ttgtagctgc tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg
     1321 aagttatggc gcgccttagc tgtgggagtg ggggtgtgga tgcggatgag ggagagtatt
     1381 attctgtcca ccagtgaatg tgttgaaagt gtggagaggc tggattcctg gaatagtgtt
     1441 gggaatcatg gttatggtat tgggtgtcat caggggaatg tcgtctgggc ttcggcgcat
     1501 tgccagggta tagtccggag ggcatgctgt cctcagcacc acctcatggg gatggatgga
     1561 ttcacactcg tggtccaggt ctgtgtgttt catctgaaga ctcatgatct cctcctcagg
     1621 tgcgtgcgta agatcattgg ttgtagttcg ttgcggacta catctcctat gtacgtcgtg
     1681 gcgccttttg tccttcttgt aatagagggc cgcgaaagcc agaatgttca ggaaaagcag
     1741 ggaagctcct acagctatgg tcacggacag ctctgtgctg tagtcccttt ggtctacgct
     1801 aaatggacta ggctgttgtt tggggtcgtc ttgcttagcg gttggaaatg cgctggtcac
     1861 aggggttgag tttttccgag ttggtcgaag ggtgatgtca gtgcttggga ccttggtggt
     1921 tgtacttgtg tattggctga tgtcattgag gttatgcaag tggggcacca gctcgagggt
     1981 accgcttccg cctccggagc ctcctcctgt gattccagca gcgttcacgt actcgtgcag
     2041 caccatgtgg tctcttccgg agcctcctcc gctgcctcca ccggtgctgt tgacaggtgt
     2101 gatgtagttg ctagggaccc agccctggcc gttcttggtc tgggcctcgc accactcgcc
     2161 gttgtggttg tagcccagga ctctcagctt ctcgcccttg gtgatgctca gggtgttgtc
     2221 gccagaggcg acgaagtcgt acagggcgac gaacaggtta gggtcgttgc ctccgaccgg
     2281 tgcgtcgccg gtgctgccgg gcacccagag cagcagcacc catagcagga gggtgtcggt
     2341 ctcaggtcca gggttctcct ccacgtctcc agcctgcttc agcaggctga agttagtagc
     2401 tccgcttcct gtagagtcca gtcccagcag aggattaggg ataggcttgc cggatccgcg
     2461 ttggtggtgg gcggcggtga agtgcagcga taagaagtcg gcgaacattt cggccgcgac
     2521 gcgctgcgcc atcggcacgg cacgcggctc gtaatgatga cagatgatca atccccatag
     2581 cgtgccgtca atgatgatcg acagcgacat cgaggcgctg acgcccatgt tccgcagaaa
     2641 ttcgagatgg cagggcgaga tgctgcgcag gtgcgcgaac gacagatcga gcgcggcgcc
     2701 ggaggcgtcg tgctcgggca cgatccggct gctgatgccg cgcgaatccg agaccacgcg
     2761 gatcgcgttc ttcaagtaca gtagccgcgc ctgctgcggg accagcgacg ccggaaagtg
     2821 ctgaccgaga aagctctcga ggtcgctgcg cttcgcctcg ccgatcacca tcccggagcc
     2881 gtcgtccgcg aagcggtaca acatcacgcg gtgatagccg agcatcgcct gcagatagcg
     2941 cggcacccgt gcggccatct cttcgagcga cttcagttct ttggtgcgcg cgatgatctg
     3001 ccgcgtcagc cgcagcggat tgtcggcctg ttcggccgcc gcaggctcga actcgatgat
     3061 cgatgtaccg tcatggcgat gcagtgagat gtcgaaggtg cggccggtca ggccgtcgcg
     3121 ccaaccgaag atcagcgccg gtcgctttgg atcggaggac tgcgccagtg cgttgcgcag
     3181 cgcatgggct tcggtctcgc cgaagtaatc ggcgagtagc tcaccgaccc gcggagtttc
     3241 gcgtccaaag aacgcgccgg cattttccgt aatgcgcgtg atccgcaccg cctgcgcgtc
     3301 gcaggctagc aggcagccgc acggctgaat gctgccgggg atgtggatcg gctcgcgatc
     3361 gcaggaggtg agatcgacct tacgcgcaga gcctcctgtc agcttggtag cctccttgtc
     3421 cttacactgc acacagccca tggtggctag aataacttcg tataaagtat cctatacgaa
     3481 gttatttgcc ttaacccaga aattatcact gttattcttt agaatggtgc aaagaataac
     3541 ttcgtataat gtatgctata cgaagttatg aattcctgca gccccgataa tcaacctctg
     3601 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     3661 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     3721 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     3781 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     3841 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     3901 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     3961 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc
     4021 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     4081 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     4141 cagacgagtc ggatctccct ttgggccgcc tccccgcatc ggggggatcc tgtgccttct
     4201 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     4261 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     4321 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     4381 agcaggcatg ctggggatgc ggtgggctct atggcggccg caggaacccc tagtgatgga
     4441 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     4501 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     4561 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     4621 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4681 acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc
     4741 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4801 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     4861 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4921 acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc
     4981 tattcttttg atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg
     5041 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     5101 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     5161 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     5221 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     5281 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     5341 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     5401 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     5461 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     5521 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     5581 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     5641 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     5701 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     5761 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5821 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5881 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     5941 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     6001 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     6061 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6121 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     6181 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     6241 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     6301 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     6361 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6421 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6481 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6541 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6601 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     6661 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6721 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6781 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6841 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6901 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6961 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7021 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     7081 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7141 tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc
     7201 cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg
     7261 agtggccaac tccatcacta ggggttcctg cggccgcacg cgtaagcttt gcaaagatgg
     7321 ataaagtttt aaacagagag gaatctttgc agctaatgga ccttctaggt cttgaaagga
     7381 gtgggaatt
//
