LOCUS       sequence-181191-        6014 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre dependent AAV expression of Ruby2 spaghetti monster FLAG 
            construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6014)
  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 6014)
  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..6014
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     promoter        441..718
                     /label=chicken beta-actin promoter
     primer_bind     997..1019
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1081..1100
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1135..1168
                     /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    1231..1264
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1280..1303)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(1307..1330)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(1334..1357)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(1550..1573)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(1580..1603)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(1616..1639)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(1646..1669)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(2174..2197)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(2201..2224)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(2228..2251)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2251..2260
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2279..2312)
                     /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(2383..2416)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2458..3046
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2511..2531)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2929..2940)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3089..3210
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3100..3119)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3154..3173
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3372..3501
                     /label=AAV2 ITR
     rep_origin      complement(3738..4326)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3818..3837)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4497..5288,5289..5357))
                     /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     5120..5139
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5358..5462)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5488..5943)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5625..5646)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5837..5856
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct
      181 aggaagagta ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      241 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      301 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      361 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      421 agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc
      481 ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat
      541 gggggcgggg gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg
      601 cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc
      661 ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg
      721 agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc
      781 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      841 gggctgtaat tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag
      901 ccttgagggg ctccgggagg gccctttgtg cggggggagc ggctcggggc tgtccgcggg
      961 gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg
     1021 gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg
     1081 gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattggat ccccataact
     1141 tcgtataaag tatcctatac gaagttatat caaaatagga agaccaatgc ttcaccatcg
     1201 acccgaattg ccaagcatca ccatcgaccc ataacttcgt ataatgtatg ctatacgaag
     1261 ttatactagt gctagcctac ttatcatcat catccttgta atcccctttg tcatcgtcgt
     1321 ctttatagtc acccttgtca tcatcgtctt tgtaatcgcc tccaccgcct cccagtcccg
     1381 cgaacttggc cacggcatgt tctctttgca cgacgaacat ctcattgtct gattcttcga
     1441 gccgctccag gcggtggtcg acggcatgaa tgcccggcat cttgatgttt cccacggttt
     1501 tctttgatct gtaggtggtc acgaacgaac aggacagatg tcctcctcct ttatcatcgt
     1561 catctttgta gtcctgctgc ttgtcgtcgt cgtccttgta gtcgccctgc tggcccttgt
     1621 catcatcgtc tttgtaatct tgttgcttat cgtcgtcatc cttgtaatct cctccgtcga
     1681 ccttcagcgc catgtgggtg tagcccctca gccctccgtc tgcggggtac atcatttcag
     1741 tattaggttc ccaacccttc gtctttttct gcatcactgg tccgttggac gggaaattca
     1801 ctccgcggac ttggacgtgg tacacgaggc acccatcctc cagcgaagta tcttgcatga
     1861 cagtcaccac acctccatct tcataccggg tcacccgttc ccaagtgaat ccttccggga
     1921 atgactgctt aaagaagtcc gggattccct tcgggtactt gatgaacgtg cgcgaccctc
     1981 ctccgaacga agtggccagg atatcgaaag cgaacggcag cggacctcct tcgatgacct
     2041 taatgcgcat ggtctgagtt cccatgtacg ggtttccctc tccctctccg gtgcacttga
     2101 actggtgacc attcacggag ccctccatca ccaccttcat ccgcatgttt tccttgatca
     2161 gtgagtttcc gcccttgtcg tcatcgtcct tatagtcgcc tttatcgtca tcatctttgt
     2221 agtcaccttt gtcgtcgtcg tccttgtagt ccatggtggc accggtggta ccactagtat
     2281 aacttcgtat aggatacttt atacgaagtt atcattggga ttcttcctat tttgatccaa
     2341 gcatcaccat cgaccctcta gtccagatct caccatcgac ccataacttc gtatagcata
     2401 cattatacga agttatgtcc ctcgaagagg ttcgaattcg atatcaagct tatcgataat
     2461 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     2521 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     2581 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     2641 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     2701 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     2761 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     2821 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     2881 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     2941 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     3001 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga
     3061 cccgggcggc cgcttcgagc agacatgata agatacattg atgagtttgg acaaaccaca
     3121 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     3181 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     3241 caggttcagg gggagatgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt
     3301 aaaatcgata aggatcttcc tagagcatgg ctacgtagat aagtagcatg gcgggttaat
     3361 cattaactac aaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     3421 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     3481 agtgagcgag cgagcgcgca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3541 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3601 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3661 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3721 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3781 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3841 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3901 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     3961 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4021 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4081 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4141 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4201 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4261 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4321 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4381 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4441 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4501 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4561 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4621 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4681 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4741 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4801 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     4861 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     4921 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     4981 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5041 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5101 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5161 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5221 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5281 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5341 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5401 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5461 cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg
     5521 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     5581 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     5641 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     5701 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     5761 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     5821 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     5881 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     5941 cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
     6001 cgctattacg ccag
//
