LOCUS       sequence-344638-        5113 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains restriction sites to clone in U6 promoter and sgRNA oligo. 
            CMV-driven mCherry fluorescent marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5113)
  AUTHORS   Romanelli SM, Lewis KT, Nishii A, Rupp AC, Li Z, Mori H, Schill RL, 
            Learman BS, Rhodes CJ, MacDougald OA
  TITLE     BAd-CRISPR: inducible gene knockout in interscapular brown adipose 
            tissue of adult mice.
  JOURNAL   J Biol Chem. 2021 Nov 11:101402. doi: 10.1016/j.jbc.2021.101402.
  PUBMED    34774798
REFERENCE   2  (bases 1 to 5113)
  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: "J Biol 
            Chem. 2021 Nov 11:101402. doi: 10.1016/j.jbc.2021.101402."
COMMENT     Sequence Label: AAV sgRNA Expression Plasmid
FEATURES             Location/Qualifiers
     source          1..5113
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        255..558
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        559..762
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     713..733
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      807..816
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             813..1523
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(958..976)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1226..1245
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    1543..2131
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1596..1616)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2014..2025)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2144..2368
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2376..2516
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2376..2505
                     /label=AAV2 ITR
     rep_origin      2591..3046
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2678..2697)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2888..2909
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3063..3082)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3182..3204
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3242..3260)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3328..3432
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3433..3501,3502..4293)
                     /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(3651..3670)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4464..5052
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4953..4972
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcag atgctgccct atccacgtgg gtggcactcg cagacaaaag
      181 gtacccaagg gcagatgctg ccctatccac gtgggtggca ctcgcagaca aaaggtaccc
      241 aagggcagat gctgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      301 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      361 ttccattgac gtcaatgagt ggagtattta cggtaaactg cccacttggc agtacatcaa
      421 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      481 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      541 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      601 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      661 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      721 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctagacgc gtcgacgaga
      781 tcgataagct tgattgattc gagctagcca ccatggtgag caagggcgag gaggataaca
      841 tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc
      901 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
      961 agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt
     1021 tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc
     1081 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg
     1141 tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc
     1201 gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     1261 cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc
     1321 tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga
     1381 agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca
     1441 acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg
     1501 gcatggacga gctgtacaag tgagaattcc tgcagccccg ataatcaacc tctggattac
     1561 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     1621 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     1681 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     1741 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     1801 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     1861 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     1921 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg
     1981 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     2041 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     2101 agtcggatct ccctttgggc cgcctccccg catcgggggg atcctgtgcc ttctagttgc
     2161 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     2221 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     2281 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     2341 catgctgggg atgcggtggg ctctatggcg gccgcaggaa cccctagtga tggagttggc
     2401 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     2461 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
     2521 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     2581 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     2641 agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc
     2701 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     2761 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     2821 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     2881 tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc gggctattct
     2941 tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa
     3001 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     3061 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     3121 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3181 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     3241 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     3301 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     3361 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     3421 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     3481 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     3541 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     3601 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     3661 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     3721 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     3781 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     3841 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     3901 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     3961 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     4021 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     4081 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     4141 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     4201 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     4261 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     4321 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     4381 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     4441 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4501 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4561 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     4621 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4681 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4741 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4801 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4861 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     4921 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     4981 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     5041 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     5101 tttgctcaca tgt
//
