LOCUS       sequence-366332-        7411 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector expressing cre-dependent mem-mScarlet, T2A, 
            Synaptophysin-EGFP driven by Dlx promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7411)
  TITLE     AAV vector using mDlx promoter
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7411)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: p226-AAV-dlx-dio-mScarlet-T2A-SYPEGFP
FEATURES             Location/Qualifiers
     source          1..7411
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(30..49)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    59..140
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     84..103
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    309..897
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             415..426
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     824..844
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(947..980)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(991..1024)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1102..1797
                     /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="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(1235..1253)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1503..1522
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1807..1860
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(2809..2811,2812..2814,2815..3528)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2809..2811,2812..2814,2815..3525)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2854..2875)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3115..3134)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3462..3483
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    3539..3572
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3583..3616
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(3671..3690)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          3673..3805
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     repeat_region   4524..4653
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(4715..5303)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4795..4814)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5474..6265,6266..6334))
                     /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     6097..6116
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6335..6439)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6507..6525
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6563..6585)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6685..6704
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6721..7176)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6858..6879)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7070..7089
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   7251..7391
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7262..7391
                     /label=AAV2 ITR
ORIGIN
        1 tgcatgcggt atcgataagc tgatacattg atgagtttgg acaaaccaca actagaatgc
       61 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
      121 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
      181 gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt atggctgatt
      241 atgatctaga gtcgcggcca gcttgatggc cgctatcaca gatctctcga gcagcgctcg
      301 gtatcgatgc ggggaggcgg cccaaaggga gatccgactc gtctgagggc gaaggcgaag
      361 acgcggaaga ggccgcagag ccggcagcag gccgcgggaa ggaaggtccg ctggattgag
      421 ggccgaaggg acgtagcaga aggacgtccc gcgcagaatc caggtggcaa cataggcgag
      481 cagccaagga aaggacgatg atttccccga caacaccacg gaattgtcag tgcccaacag
      541 ccgagcccct gtccagcagc gggcaaggca ggcggcgatg agttccgccg tggcaatagg
      601 gagggggaaa gcgaaagtcc cggaaaggag ctgacaggtg gtggcaatgc cccaaccagt
      661 gggggttgcg tcagcaaaca cagtgcacac cacgccacgt tgcctgacaa cgggccacaa
      721 ctcctcataa agagacagca accaggattt atacaaggag gagaaaatga aagccatacg
      781 ggaagcaata gcatgataca aaggcattaa agcagcgtat ccacatagcg taaaaggagc
      841 aacatagtta agaataccag tcaatctttc acaaattttg taatccagag gttgattatc
      901 gataagcttg atatcgaatt ccctgcaggg gtggtgatga tcttcgataa cttcgtatag
      961 gatactttat acgaagttat tgcgattcgg ataacttcgt atagcataca ttatacgaag
     1021 ttatcaaggc cggccgccac catgctgtgc tgtatgagaa gaaccaaaca ggttgaaaag
     1081 aatgatgagg accaaaagat catggtgagc aagggcgagg cagtgatcaa ggagttcatg
     1141 cggttcaagg tgcacatgga gggctccatg aacggccacg agttcgagat cgagggcgag
     1201 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc
     1261 cccctgccct tctcctggga catcctgtcc cctcagttca tgtacggctc cagggccttc
     1321 atcaagcacc ccgccgacat ccccgactac tataagcagt ccttccccga gggcttcaag
     1381 tgggagcgcg tgatgaactt cgaggacggc ggcgccgtga ccgtgaccca ggacacctcc
     1441 ctggaggacg gcaccctgat ctacaaggtg aagctccgcg gcaccaactt ccctcctgac
     1501 ggccccgtaa tgcagaagaa gacaatgggc tgggaagcgt ccaccgagcg gttgtacccc
     1561 gaggacggcg tgctgaaggg cgacattaag atggccctgc gcctgaagga cggcggccgc
     1621 tacctggcgg acttcaagac cacctacaag gccaagaagc ccgtgcagat gcccggcgcc
     1681 tacaacgtcg accgcaagtt ggacatcacc tcccacaacg aggactacac cgtggtggaa
     1741 cagtacgaac gctccgaggg ccgccactcc accggcggca tggacgagct gtacaaggga
     1801 agtggagagg gcagaggaag tcttctgaca tgcggagacg tggaagagaa tcccggccct
     1861 gccccaggct catctagaat ggacgtggtg aatcagctgg tggctggggg tcagttccgg
     1921 gtggtcaagg agccccttgg cttcgtgaag gtgctgcagt gggtctttgc catcttcgcc
     1981 tttgctacgt gcggcagcta caccggagag cttcggctga gcgtggagtg tgccaacaag
     2041 acggagagtg ccctcaacat cgaagtcgaa tttgagtacc cattcaggct gcaccaagtg
     2101 tactttgatg caccctcctg cgttaaaggg ggcactacca agatcttcct agttggtgac
     2161 tactcctcct cggctgaatt ctttgtcacc gtggctgtgt ttgccttcct ctactccatg
     2221 ggggccctgg ccacctacat cttcctgcag aacaagtacc gagagaacaa caaagggcca
     2281 atgatggact tcctggccac agcagtgttc gctttcatgt ggctagttag ctcatccgcc
     2341 tgggccaaag gcctgtccga tgtgaagatg gccactgacc cagagaacat tatcaaggag
     2401 atgcctatgt gccgccagac aggaaacaca tgcaaggaac tgagggaccc tgtgacttca
     2461 ggactcaaca cctcggtggt gtttggcttc ctgaacctgg tgctctgggt tggcaaccta
     2521 tggttcgtgt tcaaggagac aggctgggcc gccccattca tgcgcgcacc tccaggcgcc
     2581 ccagaaaagc aaccagctcc tggcgatgcc tacggcgatg cgggctatgg gcagggcccc
     2641 ggaggctatg ggccccagga ctcctacggg cctcagggtg gttatcaacc cgattacggg
     2701 cagccagcca gcggcggtgg cggtggctac gggcctcagg gcgactatgg gcagcaaggc
     2761 tacggccaac agggtgcgcc cacctccttc tccaatcaga tgtcgcgaat ggtgagcaag
     2821 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     2881 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     2941 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     3001 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     3061 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     3121 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     3181 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     3241 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     3301 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     3361 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     3421 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     3481 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt ataagtaagg cgcgccaaat
     3541 aacttcgtat aaagtatcct atacgaagtt attgtgattc ggataacttc gtataatgta
     3601 tgctatacga agttatcgac gtcgagtggc tactagtttc ttaagagctg taattgaact
     3661 gggagtggac acctgtggag agaaaggcaa agtggatgtc agtaagacca ataggtgcct
     3721 atcagaaacg caagagtctt ctctgtctcg acaagcccag tttctattgg tctccttaaa
     3781 cctgtcttgt aaccttgata cttacctgcc cagtgcctca cgaccaactt ctgcagctta
     3841 aggcgccgcg ctctgcttct ggaaggctgc gctcctcgac ctgcaggcta gaagcaaatg
     3901 taagcaatag atggctctgc cctgactttt atgcccagcc cgggctgcag gaattcgata
     3961 tcaagcttcc tactgtgaaa ctttgggttc gtagctcccc aggatcaatt ctgaacaaag
     4021 cctccagctg cagtgccatc caatttgaag cagacattgg ggacaattta aggtttttat
     4081 ccacaagaag gtttttttcc attctcttaa atgcagccat aattagagta atttttcatg
     4141 tagcccgctg attacagcgt ttttaccgtc aaagataatt acctgtaatt ttcttccact
     4201 tttaatacta aaaagccatc tttatttaga ttcaggaaca ggaaaggcga aacaaaagag
     4261 ggaaattatt ctgttattca tacacaaatt gcagagacgt aggacctaaa attgaaaatt
     4321 aaccaaaatt ataatgctgg aaagaatgga agaggctgca gaagtacagc tggtagtagg
     4381 gctttgctga attattcaaa ttatgttaga gaaaaagcta ccatatatcg aaaccaacac
     4441 taatttttcc ttaaaaaatt tcaccaaata tatgccaaac cactgtgagt gtatagaatt
     4501 cttaattaaa cgcgtgcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct
     4561 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggcggcct
     4621 cagtgagcga gcgagcgcgc agctgcctgc aggacatgtg agcaaaaggc cagcaaaagg
     4681 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4741 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4801 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4861 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4921 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4981 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5041 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5101 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5161 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5221 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5281 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5341 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5401 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5461 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5521 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5581 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5641 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5701 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5761 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     5821 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5881 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5941 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6001 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6061 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6121 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6181 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6241 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6301 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6361 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6421 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     6481 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc
     6541 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     6601 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     6661 ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     6721 aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     6781 tttttaacca atagaccgaa atcggcaaaa tcccttataa atcaaaagaa tagcccgaga
     6841 tagagttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     6901 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccca
     6961 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     7021 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     7081 cgaaaggagc gggcgctaag gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     7141 cacccgccgc gcttaatgcg ccgctacagg gcgcgtacta tggttgcttt gacgtatgcg
     7201 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc cctgcaggca
     7261 gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt
     7321 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
     7381 taggggttcc tgcggccgct cggtccgcac g
//
