LOCUS       sequence-212614-        6184 bp    DNA     circular SYN 15-DEC-2025
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    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6184)
  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 6184)
  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-myrmScarlet-I-P2A-post-eGRASP
FEATURES             Location/Qualifiers
     source          1..6184
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   156..285
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        301..679
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     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"
     protein_bind    417..435
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    453..471
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    489..507
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    525..543
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     588..612
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          727..859
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     842..861
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        904..922
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      926..935
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             983..1675
                     /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(1113..1131)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1381..1400
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1685..1741
                     /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             2030..2077
                     /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(2372..2383)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     misc_feature    2769..3357
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2822..2842)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3240..3251)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3370..3594
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3602..3742
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3602..3731
                     /label=AAV2 ITR
     rep_origin      3817..4272
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3904..3923)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4114..4135
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4289..4308)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4408..4430
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4468..4486)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4554..4658
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4659..4727,4728..5519)
                     /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(4877..4896)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5690..6184,1..94)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     join(6179..6184,1..14)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
       61 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
      121 gttcctggcc ttttgctggc cttttgctca catgtcctgc aggcagctgc gcgctcgctc
      181 gctcactgag gccgcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc
      241 gagcgcgcag agagggagtg gccaactcca tcactagggg ttcctgcggc cgcacgcgtt
      301 ggctttactc cctatcagtg atagagaacg tatgaagagt ttactcccta tcagtgatag
      361 agaacgtatg cagactttac tccctatcag tgatagagaa cgtataagga gtttactccc
      421 tatcagtgat agagaacgta tgaccagttt actccctatc agtgatagag aacgtatcta
      481 cagtttactc cctatcagtg atagagaacg tatatccagt ttactcccta tcagtgatag
      541 agaacgtata agctttaggc gtgtacggtg ggcgcctata aaagcagagc tcgtttagtg
      601 aaccgtcaga tcgcctggag caattccaca acacttttgt cttataccaa ctttccgtac
      661 cacttcctac cctcgtaaag ctagtcgaac ttaagctgca gaagttggtc gtgaggcact
      721 gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa actgggcttg
      781 tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac tgacatccac
      841 tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta aggctagagt
      901 acttaatacg actcactata ggctagccac catgggctgt gtgcagtgta aggacaagga
      961 ggctaccaag ctgacaggag gcgtgagcaa gggcgaggca gtgatcaagg agttcatgcg
     1021 gttcaaggtg cacatggagg gctccatgaa cggccacgag ttcgagatcg agggcgaggg
     1081 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc
     1141 cctgcccttc tcctgggaca tcctgtcccc tcagttcatg tacggctcca gggccttcat
     1201 caagcacccc gccgacatcc ccgactacta taagcagtcc ttccccgagg gcttcaagtg
     1261 ggagcgcgtg atgaacttcg aggacggcgg cgccgtgacc gtgacccagg acacctccct
     1321 ggaggacggc accctgatct acaaggtgaa gctccgcggc accaacttcc ctcctgacgg
     1381 ccccgtaatg cagaagaaga caatgggctg ggaagcgtcc accgagcggt tgtaccccga
     1441 ggacggcgtg ctgaagggcg acattaagat ggccctgcgc ctgaaggacg gcggccgcta
     1501 cctggcggac ttcaagacca cctacaaggc caagaagccc gtgcagatgc ccggcgccta
     1561 caacgtcgac cgcaagttgg acatcacctc ccacaacgag gactacaccg tggtggaaca
     1621 gtacgaacgc tccgagggcc gccactccac cggcggcatg gacgagctgt acaagggaag
     1681 cggagctact aacttcagcc tgctgaagca ggctggagac gtggaggaga accctggacc
     1741 tgagaccgac accctcctgc tatgggtgct gctgctctgg gtgcccggca gcaccggcga
     1801 cgcaccggtc ggaggcaacg accctaacct gttcgtcgcc ctgtacgact tcgtcgcctc
     1861 tggcgacaac accctgagca tcaccaaggg cgagaagctg agagtcctgg gctacaacca
     1921 caacggcgag tggtgcgagg cccagaccaa gaacggccag ggctgggtcc ctagcaacta
     1981 catcacacct gtcaacagca ccggtggagg cagcggagga ggctccggaa gagaccacat
     2041 ggtgctgcac gagtacgtga acgctgctgg aatcacagga ggaggctccg gaggcggaag
     2101 cggtaccctc gagctggtgc cccacttgca taacctcaat gacatcagcc aatacacaag
     2161 tacaaccacc aaggtcccaa gcactgacat cacccttcga ccaactcgga aaaactcaac
     2221 ccctgtgacc agcgcatttc caaccgctaa gcaagacgac cccaaacaac agcctagtcc
     2281 atttagcgta gaccaaaggg actacagcac agagctgtcc gtgaccatag ctgtaggagc
     2341 ttccctgctt ttcctgaaca ttctggcttt cgcggccctc tattacaaga aggacaaaag
     2401 gcgccacgac gtacatagga gatgtagtcc gcaacgaact acaaccaatg atcttacgca
     2461 cgcacctgag gaggagatca tgagtcttca gatgaaacac acagacctgg accacgagtg
     2521 tgaatccatc catccccatg aggtggtgct gaggacagca tgccctccgg actataccct
     2581 ggcaatgcgc cgaagcccag acgacattcc cctgatgaca cccaatacca taaccatgat
     2641 tcccaacact attccaggaa tccagcctct ccacactttc aacacattca ctggtggaca
     2701 gaataatact ctccctcatc cgcatccaca cccccactcc cacagctaag aattcctgca
     2761 gccccgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     2821 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     2881 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     2941 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     3001 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     3061 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     3121 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt
     3181 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     3241 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     3301 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc
     3361 ggggggatcc tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     3421 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     3481 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     3541 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcggccg
     3601 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     3661 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     3721 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     3781 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     3841 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     3901 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3961 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4021 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     4081 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     4141 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     4201 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     4261 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     4321 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     4381 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     4441 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     4501 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     4561 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4621 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     4681 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     4741 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     4801 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     4861 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     4921 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     4981 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5041 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5101 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     5161 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     5221 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     5281 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     5341 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     5401 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     5461 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     5521 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     5581 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     5641 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     5701 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     5761 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     5821 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     5881 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     5941 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6001 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6061 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6121 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     6181 gaaa
//
