LOCUS       Exported                6195 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  An AAV vector that expresses myristoylated mScarlet-I and 
            post-eGRASP linked by self-cleaving P2A peptide under the tetO 
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-TIWB-myrmScarlet-I-P2A-post-eGRASP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6195)
  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 6195)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6195
                     /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..1531
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     primer_bind     complement(969..987)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1237..1256
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1541..1597
                     /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             1886..1933
                     /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(2228..2239)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     misc_feature    2625..3213
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2678..2698)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3096..3107)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3226..3450
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3458..3598
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3458..3587
                     /label=AAV2 ITR
     rep_origin      3673..4128
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3760..3779)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3970..3991
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4145..4164)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4264..4286
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4324..4342)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4410..4514
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4515..5375
                     /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(4733..4752)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5546..6134
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6035..6054
                     /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 caggaggcgt
      841 gagcaagggc gaggcagtga tcaaggagtt catgcggttc aaggtgcaca tggagggctc
      901 catgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac
      961 ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttctcct gggacatcct
     1021 gtcccctcag ttcatgtacg gctccagggc cttcatcaag caccccgccg acatccccga
     1081 ctactataag cagtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga
     1141 cggcggcgcc gtgaccgtga cccaggacac ctccctggag gacggcaccc tgatctacaa
     1201 ggtgaagctc cgcggcacca acttccctcc tgacggcccc gtaatgcaga agaagacaat
     1261 gggctgggaa gcgtccaccg agcggttgta ccccgaggac ggcgtgctga agggcgacat
     1321 taagatggcc ctgcgcctga aggacggcgg ccgctacctg gcggacttca agaccaccta
     1381 caaggccaag aagcccgtgc agatgcccgg cgcctacaac gtcgaccgca agttggacat
     1441 cacctcccac aacgaggact acaccgtggt ggaacagtac gaacgctccg agggccgcca
     1501 ctccaccggc ggcatggacg agctgtacaa gggaagcgga gctactaact tcagcctgct
     1561 gaagcaggct ggagacgtgg aggagaaccc tggacctgag accgacaccc tcctgctatg
     1621 ggtgctgctg ctctgggtgc ccggcagcac cggcgacgca ccggtcggag gcaacgaccc
     1681 taacctgttc gtcgccctgt acgacttcgt cgcctctggc gacaacaccc tgagcatcac
     1741 caagggcgag aagctgagag tcctgggcta caaccacaac ggcgagtggt gcgaggccca
     1801 gaccaagaac ggccagggct gggtccctag caactacatc acacctgtca acagcaccgg
     1861 tggaggcagc ggaggaggct ccggaagaga ccacatggtg ctgcacgagt acgtgaacgc
     1921 tgctggaatc acaggaggag gctccggagg cggaagcggt accctcgagc tggtgcccca
     1981 cttgcataac ctcaatgaca tcagccaata cacaagtaca accaccaagg tcccaagcac
     2041 tgacatcacc cttcgaccaa ctcggaaaaa ctcaacccct gtgaccagcg catttccaac
     2101 cgctaagcaa gacgacccca aacaacagcc tagtccattt agcgtagacc aaagggacta
     2161 cagcacagag ctgtccgtga ccatagctgt aggagcttcc ctgcttttcc tgaacattct
     2221 ggctttcgcg gccctctatt acaagaagga caaaaggcgc cacgacgtac ataggagatg
     2281 tagtccgcaa cgaactacaa ccaatgatct tacgcacgca cctgaggagg agatcatgag
     2341 tcttcagatg aaacacacag acctggacca cgagtgtgaa tccatccatc cccatgaggt
     2401 ggtgctgagg acagcatgcc ctccggacta taccctggca atgcgccgaa gcccagacga
     2461 cattcccctg atgacaccca ataccataac catgattccc aacactattc caggaatcca
     2521 gcctctccac actttcaaca cattcactgg tggacagaat aatactctcc ctcatccgca
     2581 tccacacccc cactcccaca gctaagaatt cctgcagccc cgataatcaa cctctggatt
     2641 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     2701 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     2761 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     2821 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     2881 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     2941 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     3001 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
     3061 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     3121 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     3181 cgagtcggat ctccctttgg gccgcctccc cgcatcgggg ggatcctgtg ccttctagtt
     3241 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     3301 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     3361 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     3421 ggcatgctgg ggatgcggtg ggctctatgg cggccgcagg aacccctagt gatggagttg
     3481 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     3541 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     3601 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     3661 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3721 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3781 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3841 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     3901 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3961 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcgggctatt
     4021 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     4081 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     4141 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     4201 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4261 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     4321 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     4381 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4441 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4501 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4561 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     4621 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     4681 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     4741 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     4801 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     4861 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     4921 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     4981 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5041 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     5101 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     5161 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5221 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5281 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5341 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5401 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5461 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5521 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5581 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5641 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     5701 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5761 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5821 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5881 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5941 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6001 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6061 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6121 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6181 cttttgctca catgt
//
