LOCUS       sequence-189883-        5922 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Cre dependent AAV expression of Ruby2 spaghetti monster FLAG 
            construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5922)
  AUTHORS   Viswanathan S, Williams ME, Bloss EB, Stasevich TJ, Speer CM, Nern 
            A, Pfeiffer BD, Hooks BM, Li WP, English BP, Tian T, Henry GL, 
            Macklin JJ, Patel R, Gerfen CR, Zhuang X, Wang Y, Rubin GM, Looger 
            LL
  TITLE     High-performance probes for light and electron microscopy.
  JOURNAL   Nat Methods. 2015 Jun;12(6):568-76. doi: 10.1038/nmeth.3365. Epub 
            2015 Apr 27.
  PUBMED    25915120
REFERENCE   2  (bases 1 to 5922)
  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: 
            "10.1038/nmeth.3365"; journalName: "Nat Methods"; date: "2015-06"; 
            volume: "12"; issue: "6"; pages: "568-76"
COMMENT     Sequence Label: pAAV.CAG.Flex.Ruby2sm-Flag.WPRE.SV40
FEATURES             Location/Qualifiers
     source          1..5922
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     351..373
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     435..454
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    489..522
                     /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    585..618
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(634..657)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(661..684)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(688..711)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(904..927)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(934..957)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(970..993)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(1000..1023)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(1528..1551)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(1555..1578)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(1582..1605)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      1605..1614
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(1633..1666)
                     /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(1737..1770)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    1812..2400
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1865..1885)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2283..2294)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2443..2564
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2454..2473)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2508..2527
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   2726..2855
                     /label=AAV2 ITR
     rep_origin      complement(3092..3680)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3172..3191)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3851..4642,4643..4711))
                     /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     4474..4493
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4712..4816)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4842..5297)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4979..5000)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5191..5210
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   5369..5498
                     /label=AAV2 ITR
ORIGIN
        1 ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc
       61 gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc
      121 tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac
      181 ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg
      241 tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg
      301 gggctgtccg cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt
      361 ctggcgtgtg accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt
      421 cctacagctc ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt
      481 ggatccccat aacttcgtat aaagtatcct atacgaagtt atatcaaaat aggaagacca
      541 atgcttcacc atcgacccga attgccaagc atcaccatcg acccataact tcgtataatg
      601 tatgctatac gaagttatac tagtgctagc ctacttatca tcatcatcct tgtaatcccc
      661 tttgtcatcg tcgtctttat agtcaccctt gtcatcatcg tctttgtaat cgcctccacc
      721 gcctcccagt cccgcgaact tggccacggc atgttctctt tgcacgacga acatctcatt
      781 gtctgattct tcgagccgct ccaggcggtg gtcgacggca tgaatgcccg gcatcttgat
      841 gtttcccacg gttttctttg atctgtaggt ggtcacgaac gaacaggaca gatgtcctcc
      901 tcctttatca tcgtcatctt tgtagtcctg ctgcttgtcg tcgtcgtcct tgtagtcgcc
      961 ctgctggccc ttgtcatcat cgtctttgta atcttgttgc ttatcgtcgt catccttgta
     1021 atctcctccg tcgaccttca gcgccatgtg ggtgtagccc ctcagccctc cgtctgcggg
     1081 gtacatcatt tcagtattag gttcccaacc cttcgtcttt ttctgcatca ctggtccgtt
     1141 ggacgggaaa ttcactccgc ggacttggac gtggtacacg aggcacccat cctccagcga
     1201 agtatcttgc atgacagtca ccacacctcc atcttcatac cgggtcaccc gttcccaagt
     1261 gaatccttcc gggaatgact gcttaaagaa gtccgggatt cccttcgggt acttgatgaa
     1321 cgtgcgcgac cctcctccga acgaagtggc caggatatcg aaagcgaacg gcagcggacc
     1381 tccttcgatg accttaatgc gcatggtctg agttcccatg tacgggtttc cctctccctc
     1441 tccggtgcac ttgaactggt gaccattcac ggagccctcc atcaccacct tcatccgcat
     1501 gttttccttg atcagtgagt ttccgccctt gtcgtcatcg tccttatagt cgcctttatc
     1561 gtcatcatct ttgtagtcac ctttgtcgtc gtcgtccttg tagtccatgg tggcaccggt
     1621 ggtaccacta gtataacttc gtataggata ctttatacga agttatcatt gggattcttc
     1681 ctattttgat ccaagcatca ccatcgaccc tctagtccag atctcaccat cgacccataa
     1741 cttcgtatag catacattat acgaagttat gtccctcgaa gaggttcgaa ttcgatatca
     1801 agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     1861 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     1921 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     1981 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     2041 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     2101 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     2161 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     2221 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     2281 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     2341 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     2401 atcgataccg tcgacccggg cggccgcttc gagcagacat gataagatac attgatgagt
     2461 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     2521 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     2581 ttcattttat gtttcaggtt cagggggaga tgtgggaggt tttttaaagc aagtaaaacc
     2641 tctacaaatg tggtaaaatc gataaggatc ttcctagagc atggctacgt agataagtag
     2701 catggcgggt taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct
     2761 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     2821 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgca ttaatgaatc ggccaacgcg
     2881 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     2941 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     3001 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     3061 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3121 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3181 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3241 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     3301 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3361 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     3421 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     3481 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     3541 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3601 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3661 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     3721 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     3781 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     3841 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3901 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3961 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     4021 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     4081 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     4141 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     4201 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     4261 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     4321 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     4381 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     4441 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     4501 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     4561 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     4621 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     4681 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     4741 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4801 aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat
     4861 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     4921 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     4981 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     5041 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     5101 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     5161 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     5221 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     5281 gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc
     5341 ggtgcgggcc tcttcgctat tacgccagct gcgcgctcgc tcgctcactg aggccgcccg
     5401 ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg
     5461 cagagaggga gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc
     5521 atgctactta tctacgtagc catgctctag gaagagtacc attgacgtca ataatgacgt
     5581 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     5641 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     5701 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     5761 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc
     5821 ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat
     5881 ttatttttta attattttgt gcagcgatgg gggcgggggg gg
//
