LOCUS       sequence-190984-        5654 bp    DNA     linear   UNA 14-APR-2025
DEFINITION  Can be used to generate AAV virus that will express mRuby3 in the 
            presence of Cre.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5654)
  AUTHORS   Rylan Larsen
  TITLE     AAV red fluorescent protein plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5654)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: AAV pCAG-FLEX-mRuby3-WPRE
FEATURES             Location/Qualifiers
     source          1..5654
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     320..342
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     404..423
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(500..533)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(544..577)
                     /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)."
     CDS             complement(591..1301)
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD
                     IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV
                     AKYSNLGGGMDELYK"
     protein_bind    1319..1352
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1363..1396
                     /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)."
     misc_feature    1420..2008
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1473..1493)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1891..1902)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2043..2060)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2049..2256
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2279..2419
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2279..2408
                     /label=AAV2 ITR
     rep_origin      2494..2949
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2581..2600)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2791..2812
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2966..2985)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3085..3107
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3145..3163)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3231..3335
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3336..3404,3405..4196)
                     /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(3554..3573)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4367..4955
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4856..4875
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5017..5146
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        5239..5542
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ggctcggggg aggggcgcgg cggcccccgg agcgccggcg gctgtcgagg cgcggcgagc
       61 cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc tttgtcccaa
      121 atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc cctctagcgg gcgcggggcg
      181 aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg gccttcgtgc gtcgccgcgc
      241 cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc ggggggacgg ctgccttcgg
      301 gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc tagagcctct
      361 gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt gctggttatt
      421 gtgctgtctc atcattttgg caaagaattg atttgatacc gcgggccccg cgtggacggt
      481 acccttcagg tcgacgtcga taacttcgta tagcatacat tatacgaagt tatccgaatc
      541 gcaataactt cgtataggat actttatacg aagttatttg gcgcgcctga cttgtacagc
      601 tcgtccatgc caccaccaag gttgctgtat ttggcaactg ccacttctct ttgcactacg
      661 taggtttcat tgtcactctc ctcgatcctt tccaggcggt gatcaacggc atggacaccg
      721 ggcatcttga tgttcccgac ggtctttttt gacctgtaag ttgtcacgaa gttgcagtgc
      781 agatggccac caccatcaac tttcagtgcg atgtcagtgt atcctctcag accaccatct
      841 gctggataca tcatctctgt attaggctcc caacccttgg tcttcttctg catcacggga
      901 ccattggagg gaaagtttac ccctctgacc ttgacgttgt agacgagctc gccatcctca
      961 aggctggtgt cctgcgtgac ggtgacgact ccaccatctt cgtatctcgt aactctttcc
     1021 caagtaaaac cctcaggaaa ggactgttta aagaaatcag ggatgtcggc cgggtacttg
     1081 ataaaggtac ggctgccata catgaacgac gtggcaagaa tgtcaaaggc aaatggcagg
     1141 ggtcctccct cgatgacttt gatcctcatg gtttgcactc cctcgtacgg tctgccttct
     1201 ccttcacctg tgcatttgaa ttggtggccg ttgaccgaac cttccatgac caccttcata
     1261 cgcatatttt ccttgatcag ctcttcgccc ttagacacca tgccaccggc cggccttgat
     1321 aacttcgtat aatgtatgct atacgaagtt atccgaatcg caataacttc gtataaagta
     1381 tcctatacga agttatcgaa gatcatcaag cttatcgata atcaacctct ggattacaaa
     1441 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     1501 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     1561 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     1621 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     1681 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     1741 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     1801 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctatgttgc cacctggatt
     1861 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     1921 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
     1981 cggatctccc tttgggccgc ctccccgcat cgataccgag cgctgctcga gagatcgatc
     2041 tgcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2101 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2161 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2221 ggaggattgg gaagacaata gcaggcatgc tggggacacg tgcggaccga gcggccgcag
     2281 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     2341 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     2401 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     2461 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     2521 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
     2581 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2641 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     2701 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     2761 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     2821 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt
     2881 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     2941 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     3001 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3061 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3121 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     3181 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3241 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3301 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     3361 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     3421 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     3481 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     3541 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     3601 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     3661 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     3721 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     3781 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     3841 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     3901 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     3961 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     4021 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     4081 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     4141 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     4201 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     4261 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4321 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4381 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4441 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4501 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4561 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4621 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4681 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4741 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4801 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4861 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4921 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4981 ggttcctggc cttttgctgg ccttttgctc acatgtcctg caggcagctg cgcgctcgct
     5041 cgctcactga ggccgcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag
     5101 cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcgg ccgcacgcga
     5161 aacaattctg caggaatcta gttattaata gtaatcaatt acggggtcat tagttcatag
     5221 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     5281 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     5341 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     5401 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     5461 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     5521 attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat
     5581 ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc
     5641 gatgggggcg gggg
//
