LOCUS       Exported                6228 bp ds-DNA     circular SYN 30-MAY-2023
DEFINITION  An AAV vector that expresses myristoylated TagRFP-T and post-eGRASP 
            linked by self-cleaving P2A peptide under the tetO promoter..
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-TIWB-myrTagRFP-T-P2A-post-eGRASP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6228)
  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 6228)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 30, 2023 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6228
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     protein_bind    165..183
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    201..219
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    237..255
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    273..291
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    309..327
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    345..363
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    381..399
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     444..468
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          583..715
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     698..717
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        760..778
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      782..791
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             839..1564
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="SKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRI
                     KVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVL
                     TATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDMA
                     LKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVAR
                     YCDLPSKLGHKLNGMDELYK"
     CDS             1574..1630
                     /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             1919..1966
                     /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(2261..2272)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     misc_feature    2658..3246
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2711..2731)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3129..3140)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3259..3483
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3491..3631
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3491..3620
                     /label=AAV2 ITR
     rep_origin      3706..4161
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3793..3812)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4003..4024
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4178..4197)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4297..4319
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4357..4375)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4443..4547
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4548..5408
                     /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(4766..4785)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5579..6167
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6068..6087
                     /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 cgcgttggct ttactcccta tcagtgatag
      181 agaacgtatg aagagtttac tccctatcag tgatagagaa cgtatgcaga ctttactccc
      241 tatcagtgat agagaacgta taaggagttt actccctatc agtgatagag aacgtatgac
      301 cagtttactc cctatcagtg atagagaacg tatctacagt ttactcccta tcagtgatag
      361 agaacgtata tccagtttac tccctatcag tgatagagaa cgtataagct ttaggcgtgt
      421 acggtgggcg cctataaaag cagagctcgt ttagtgaacc gtcagatcgc ctggagcaat
      481 tccacaacac ttttgtctta taccaacttt ccgtaccact tcctaccctc gtaaagctag
      541 tcgaacttaa gctgcagaag ttggtcgtga ggcactgggc aggtaagtat caaggttaca
      601 agacaggttt aaggagacca atagaaactg ggcttgtcga gacagagaag actcttgcgt
      661 ttctgatagg cacctattgg tcttactgac atccactttg cctttctctc cacaggtgtc
      721 cactcccagt tcaattacag ctcttaaggc tagagtactt aatacgactc actataggct
      781 agccaccatg ggctgtgtgc agtgtaagga caaggaggct accaagctga caggaggctc
      841 taagggcgaa gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt
      901 gaacaaccac cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca
      961 gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc
     1021 taccagcttc atgtacggca gcagaacctt catcaaccac acccagggca tccccgactt
     1081 ctttaagcag tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg
     1141 gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt
     1201 caagatcaga ggggtgaact tcccatccaa cggccctgtg atgcagaaga aaacactcgg
     1261 ctgggaggcc aacaccgaga tgctgtaccc cgctgacggc ggcctggaag gcagaaccga
     1321 catggccctg aagctcgtgg gcgggggcca cctgatctgc aacttcaaga ccacatacag
     1381 atccaagaaa cccgctaaga acctcaagat gcccggcgtc tactatgtgg accacagact
     1441 ggaaagaatc aaggaggccg acaaagagac ctacgtcgag cagcacgagg tggctgtggc
     1501 cagatactgc gacctcccta gcaaactggg gcacaaactt aatggcatgg acgagctgta
     1561 caagggaagc ggagctacta acttcagcct gctgaagcag gctggagacg tggaggagaa
     1621 ccctggacct gagaccgaca ccctcctgct atgggtgctg ctgctctggg tgcccggcag
     1681 caccggcgac gcaccggtcg gaggcaacga ccctaacctg ttcgtcgccc tgtacgactt
     1741 cgtcgcctct ggcgacaaca ccctgagcat caccaagggc gagaagctga gagtcctggg
     1801 ctacaaccac aacggcgagt ggtgcgaggc ccagaccaag aacggccagg gctgggtccc
     1861 tagcaactac atcacacctg tcaacagcac cggtggaggc agcggaggag gctccggaag
     1921 agaccacatg gtgctgcacg agtacgtgaa cgctgctgga atcacaggag gaggctccgg
     1981 aggcggaagc ggtaccctcg agctggtgcc ccacttgcat aacctcaatg acatcagcca
     2041 atacacaagt acaaccacca aggtcccaag cactgacatc acccttcgac caactcggaa
     2101 aaactcaacc cctgtgacca gcgcatttcc aaccgctaag caagacgacc ccaaacaaca
     2161 gcctagtcca tttagcgtag accaaaggga ctacagcaca gagctgtccg tgaccatagc
     2221 tgtaggagct tccctgcttt tcctgaacat tctggctttc gcggccctct attacaagaa
     2281 ggacaaaagg cgccacgacg tacataggag atgtagtccg caacgaacta caaccaatga
     2341 tcttacgcac gcacctgagg aggagatcat gagtcttcag atgaaacaca cagacctgga
     2401 ccacgagtgt gaatccatcc atccccatga ggtggtgctg aggacagcat gccctccgga
     2461 ctataccctg gcaatgcgcc gaagcccaga cgacattccc ctgatgacac ccaataccat
     2521 aaccatgatt cccaacacta ttccaggaat ccagcctctc cacactttca acacattcac
     2581 tggtggacag aataatactc tccctcatcc gcatccacac ccccactccc acagctaaga
     2641 attcctgcag ccccgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     2701 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     2761 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     2821 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     2881 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     2941 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     3001 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt
     3061 cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
     3121 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     3181 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     3241 ccccgcatcg gggggatcct gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     3301 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     3361 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     3421 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     3481 tggcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     3541 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     3601 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
     3661 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     3721 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3781 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3841 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3901 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     3961 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     4021 ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg attttgccga
     4081 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     4141 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     4201 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4261 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4321 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     4381 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     4441 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4501 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     4561 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     4621 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     4681 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     4741 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     4801 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     4861 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     4921 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     4981 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     5041 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     5101 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     5161 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     5221 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     5281 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     5341 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     5401 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     5461 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     5521 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     5581 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     5641 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     5701 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     5761 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     5821 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     5881 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     5941 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6001 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     6061 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     6121 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     6181 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
