LOCUS       sequence-212219-        6217 bp    DNA     circular SYN 15-DEC-2025
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    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6217)
  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 6217)
  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-TIWB-myrTagRFP-T-P2A-post-eGRASP
FEATURES             Location/Qualifiers
     source          1..6217
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          1..133
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     116..135
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        178..196
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      200..209
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             257..982
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="SKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRI
                     KVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVL
                     TATQDTSFQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDMA
                     LKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVAR
                     YCDLPSKLGHKLNGMDELYK"
     CDS             992..1048
                     /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             1337..1384
                     /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(1679..1690)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     misc_feature    2076..2664
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2129..2149)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2547..2558)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2677..2901
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2909..3049
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2909..3038
                     /label=AAV2 ITR
     rep_origin      3124..3579
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3211..3230)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3421..3442
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3596..3615)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3715..3737
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3775..3793)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3861..3965
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3966..4034,4035..4826)
                     /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(4184..4203)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4997..5585
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5486..5505
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5647..5776
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        5792..6170
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    5800..5818
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5836..5854
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5872..5890
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5908..5926
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5944..5962
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5980..5998
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6016..6034
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     6079..6103
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
ORIGIN
        1 gtaagtatca aggttacaag acaggtttaa ggagaccaat agaaactggg cttgtcgaga
       61 cagagaagac tcttgcgttt ctgataggca cctattggtc ttactgacat ccactttgcc
      121 tttctctcca caggtgtcca ctcccagttc aattacagct cttaaggcta gagtacttaa
      181 tacgactcac tataggctag ccaccatggg ctgtgtgcag tgtaaggaca aggaggctac
      241 caagctgaca ggaggctcta agggcgaaga gctgattaag gagaacatgc acatgaagct
      301 gtacatggag ggcaccgtga acaaccacca cttcaagtgc acatccgagg gcgaaggcaa
      361 gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt
      421 cgccttcgac atcctggcta ccagcttcat gtacggcagc agaaccttca tcaaccacac
      481 ccagggcatc cccgacttct ttaagcagtc cttccctgag ggcttcacat gggagagagt
      541 caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagct tccaggacgg
      601 ctgcctcatc tacaacgtca agatcagagg ggtgaacttc ccatccaacg gccctgtgat
      661 gcagaagaaa acactcggct gggaggccaa caccgagatg ctgtaccccg ctgacggcgg
      721 cctggaaggc agaaccgaca tggccctgaa gctcgtgggc gggggccacc tgatctgcaa
      781 cttcaagacc acatacagat ccaagaaacc cgctaagaac ctcaagatgc ccggcgtcta
      841 ctatgtggac cacagactgg aaagaatcaa ggaggccgac aaagagacct acgtcgagca
      901 gcacgaggtg gctgtggcca gatactgcga cctccctagc aaactggggc acaaacttaa
      961 tggcatggac gagctgtaca agggaagcgg agctactaac ttcagcctgc tgaagcaggc
     1021 tggagacgtg gaggagaacc ctggacctga gaccgacacc ctcctgctat gggtgctgct
     1081 gctctgggtg cccggcagca ccggcgacgc accggtcgga ggcaacgacc ctaacctgtt
     1141 cgtcgccctg tacgacttcg tcgcctctgg cgacaacacc ctgagcatca ccaagggcga
     1201 gaagctgaga gtcctgggct acaaccacaa cggcgagtgg tgcgaggccc agaccaagaa
     1261 cggccagggc tgggtcccta gcaactacat cacacctgtc aacagcaccg gtggaggcag
     1321 cggaggaggc tccggaagag accacatggt gctgcacgag tacgtgaacg ctgctggaat
     1381 cacaggagga ggctccggag gcggaagcgg taccctcgag ctggtgcccc acttgcataa
     1441 cctcaatgac atcagccaat acacaagtac aaccaccaag gtcccaagca ctgacatcac
     1501 ccttcgacca actcggaaaa actcaacccc tgtgaccagc gcatttccaa ccgctaagca
     1561 agacgacccc aaacaacagc ctagtccatt tagcgtagac caaagggact acagcacaga
     1621 gctgtccgtg accatagctg taggagcttc cctgcttttc ctgaacattc tggctttcgc
     1681 ggccctctat tacaagaagg acaaaaggcg ccacgacgta cataggagat gtagtccgca
     1741 acgaactaca accaatgatc ttacgcacgc acctgaggag gagatcatga gtcttcagat
     1801 gaaacacaca gacctggacc acgagtgtga atccatccat ccccatgagg tggtgctgag
     1861 gacagcatgc cctccggact ataccctggc aatgcgccga agcccagacg acattcccct
     1921 gatgacaccc aataccataa ccatgattcc caacactatt ccaggaatcc agcctctcca
     1981 cactttcaac acattcactg gtggacagaa taatactctc cctcatccgc atccacaccc
     2041 ccactcccac agctaagaat tcctgcagcc ccgataatca acctctggat tacaaaattt
     2101 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     2161 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     2221 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     2281 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     2341 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     2401 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     2461 tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc
     2521 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     2581 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     2641 tctccctttg ggccgcctcc ccgcatcggg gggatcctgt gccttctagt tgccagccat
     2701 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     2761 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     2821 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     2881 gggatgcggt gggctctatg gcggccgcag gaacccctag tgatggagtt ggccactccc
     2941 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     3001 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg
     3061 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     3121 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3181 ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3241 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3301 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     3361 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3421 gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt
     3481 tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat
     3541 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     3601 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     3661 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     3721 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     3781 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     3841 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     3901 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     3961 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     4021 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     4081 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     4141 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     4201 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     4261 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     4321 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     4381 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     4441 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     4501 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     4561 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     4621 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     4681 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     4741 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     4801 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     4861 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     4921 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     4981 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     5041 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     5101 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     5161 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     5221 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     5281 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     5341 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     5401 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     5461 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     5521 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     5581 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     5641 acatgtcctg caggcagctg cgcgctcgct cgctcactga ggccgcccgg gcgtcgggcg
     5701 acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc
     5761 atcactaggg gttcctgcgg ccgcacgcgt tggctttact ccctatcagt gatagagaac
     5821 gtatgaagag tttactccct atcagtgata gagaacgtat gcagacttta ctccctatca
     5881 gtgatagaga acgtataagg agtttactcc ctatcagtga tagagaacgt atgaccagtt
     5941 tactccctat cagtgataga gaacgtatct acagtttact ccctatcagt gatagagaac
     6001 gtatatccag tttactccct atcagtgata gagaacgtat aagctttagg cgtgtacggt
     6061 gggcgcctat aaaagcagag ctcgtttagt gaaccgtcag atcgcctgga gcaattccac
     6121 aacacttttg tcttatacca actttccgta ccacttccta ccctcgtaaa gctagtcgaa
     6181 cttaagctgc agaagttggt cgtgaggcac tgggcag
//
