LOCUS       sequence-189951-        5911 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of Ruby2 spaghetti monster FLAG construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5911)
  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 5911)
  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.Ruby2sm-Flag.WPRE.SV40
FEATURES             Location/Qualifiers
     source          1..5911
                     /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"
     regulatory      1144..1153
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1153..1176
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1180..1203
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1207..1230
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1735..1758
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1765..1788
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1801..1824
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1831..1854
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2047..2070
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2074..2097
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2101..2124
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    2152..2740
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2205..2225)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2623..2634)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2783..2904
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2794..2813)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2848..2867
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3066..3195
                     /label=AAV2 ITR
     rep_origin      3267..3722
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3354..3373)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3564..3585
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3748..3852
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3853..3921,3922..4713)
                     /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(4071..4090)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4884..5472
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5373..5392
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    5760..5781
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5796..5801,5802..5819,5820..5826)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5834..5850
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     5839..5861
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5858..5874
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5858..5874
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
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 ccgcggccgc
     1141 gccgccacca tggactacaa ggacgacgac gacaaaggtg actacaaaga tgatgacgat
     1201 aaaggcgact ataaggacga tgacgacaag ggcggaaact cactgatcaa ggaaaacatg
     1261 cggatgaagg tggtgatgga gggctccgtg aatggtcacc agttcaagtg caccggagag
     1321 ggagagggaa acccgtacat gggaactcag accatgcgca ttaaggtcat cgaaggaggt
     1381 ccgctgccgt tcgctttcga tatcctggcc acttcgttcg gaggagggtc gcgcacgttc
     1441 atcaagtacc cgaagggaat cccggacttc tttaagcagt cattcccgga aggattcact
     1501 tgggaacggg tgacccggta tgaagatgga ggtgtggtga ctgtcatgca agatacttcg
     1561 ctggaggatg ggtgcctcgt gtaccacgtc caagtccgcg gagtgaattt cccgtccaac
     1621 ggaccagtga tgcagaaaaa gacgaagggt tgggaaccta atactgaaat gatgtacccc
     1681 gcagacggag ggctgagggg ctacacccac atggcgctga aggtcgacgg aggagattac
     1741 aaggatgacg acgataagca acaagattac aaagacgatg atgacaaggg ccagcagggc
     1801 gactacaagg acgacgacga caagcagcag gactacaaag atgacgatga taaaggagga
     1861 ggacatctgt cctgttcgtt cgtgaccacc tacagatcaa agaaaaccgt gggaaacatc
     1921 aagatgccgg gcattcatgc cgtcgaccac cgcctggagc ggctcgaaga atcagacaat
     1981 gagatgttcg tcgtgcaaag agaacatgcc gtggccaagt tcgcgggact gggaggcggt
     2041 ggaggcgatt acaaagacga tgatgacaag ggtgactata aagacgacga tgacaaaggg
     2101 gattacaagg atgatgatga taagtagacc ggtgaattca agcttatcga taatcaacct
     2161 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     2221 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     2281 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     2341 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     2401 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     2461 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     2521 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
     2581 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     2641 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     2701 cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg tcgacccggg
     2761 cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga
     2821 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     2881 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     2941 cagggggaga tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc
     3001 gataaggatc ttcctagagc atggctacgt agataagtag catggcgggt taatcattaa
     3061 ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     3121 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     3181 cgagcgagcg cgcagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     3241 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     3301 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3361 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3421 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3481 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3541 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3601 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3661 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     3721 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     3781 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     3841 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     3901 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     3961 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     4021 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     4081 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     4141 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     4201 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     4261 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     4321 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     4381 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     4441 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     4501 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     4561 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     4621 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     4681 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     4741 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     4801 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     4861 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4921 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4981 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     5041 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     5101 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     5161 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     5221 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     5281 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     5341 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     5401 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     5461 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     5521 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     5581 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     5641 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     5701 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     5761 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     5821 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     5881 tacgccagat ttaattaagg ccttaattag g
//
