LOCUS       sequence-341002-        7252 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Actb sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7252)
  AUTHORS   Zhong H, Ceballos CC, Massengill CI, Muniak MA, Ma L, Qin M, Petrie 
            SK, Mao T
  TITLE     High-fidelity, efficient, and reversible labeling of endogenous 
            proteins using CRISPR-based designer exon insertion.
  JOURNAL   Elife. 2021 Jun 8;10. pii: 64911. doi: 10.7554/eLife.64911.
  PUBMED    34100715
REFERENCE   2  (bases 1 to 7252)
  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: "Elife."; 
            date: "2021-06-8"; volume: "10. pii: 64911. doi"; pages: " 
            10.7554/eLife.64911"
COMMENT     Sequence Label: pAAV_ACTB CRISPIE
FEATURES             Location/Qualifiers
     source          1..7252
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   7..136
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     complement(175..194)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(176..194)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        201..449
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        470..545
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(630..647)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        655..903
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        924..999
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1088..1106)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     regulatory      1368..1377
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1374..1376,1377..1379,1380..2090)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1419..1440)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1680..1699)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2027..2048
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        2662..2939
                     /label=chicken beta-actin promoter
     primer_bind     3218..3240
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3302..3321
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      3360..3369
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3366..4076
                     /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"
     primer_bind     complement(4141..4161)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4521..4661
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4521..4650
                     /label=AAV2 ITR
     rep_origin      4736..5191
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4823..4842)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5033..5054
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5208..5227)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5327..5349
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5387..5405)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5473..5577
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5578..5646,5647..6438)
                     /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(5796..5815)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6609..7197
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7098..7117
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acatgtcctg caggcagctg cgcgctcgct cgctcactga ggccgcccgg gcgtcgggcg
       61 acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc
      121 atcactaggg gttcctgcgg ccgcagatgc tgcccatgct ctaggaagat cgtaccctat
      181 agtgagtcgt attagaattc gagggcctat ttcccatgat tccttcatat ttgcatatac
      241 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
      301 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
      361 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
      421 tatatatctt gtggaaagga cgaaacaccg cctacactgc gccgacgggg ttttagagct
      481 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
      541 ggtgcttttt tgttttagag ctagaaatag caagttaaaa taaggctagt ccgtttttag
      601 cgcgtgcgcc aattctgcag acaaatggcc tatagtgtca cctaaatcgg taccgagggc
      661 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
      721 ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
      781 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
      841 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac
      901 accgcgtatg gcaagcatag ccggttttag agctagaaat agcaagttaa aataaggcta
      961 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgtttt agagctagaa
     1021 atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg cgccaattct gcagacaaat
     1081 ggctctccct ttagtgaggg ttaattagaa ctagtgcgta tggcaagcat agccggggcg
     1141 gccgcgcgta tggcaagcat agccggggtt ccgcgtatgg caagcatagc cggggaattc
     1201 gacgcttccg agtacggtac aggagcgctg acgcttccga gtacggtaca ggcaaattgc
     1261 tccagaaggg tgagaaggct agagatgtgg gagaactggg tggaaggcag aatcgaccac
     1321 gtctaaggtt gtgggtttac tgtgtcttac agttgaagga aaccggtgcc accatggtga
     1381 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     1441 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     1501 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     1561 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     1621 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     1681 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     1741 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     1801 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     1861 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     1921 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     1981 gcacccagtc caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     2041 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag tccggaggta
     2101 gtgctggtgg caccggactc agatctggca gcggtggagg cagcgcaggt cctgccgcag
     2161 taaggattct ttcttcctga atctctgggg tgcattaagg gtattgcctt agtagagggg
     2221 catgccccag gggagtggag gtggggtgag ggtggaggac ctgtaccgta ctcggaagcg
     2281 tcggatcccc tgtaccgtac tcggaagcgt cggtaccccc cggctatgct tgccatacgc
     2341 ttccccccgg ctatgcttgc catacgcgat atccccggct atgcttgcca tacgcaagct
     2401 tgaagatcgt accattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     2461 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     2521 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     2581 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     2641 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct
     2701 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga
     2761 tgggggcggg gggggggggg gggcgcgcgc caggcggggc ggggcggggc grggggcggg
     2821 gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc
     2881 cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg
     2941 gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc
     3001 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc
     3061 cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa
     3121 gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ctgtccgcgg
     3181 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     3241 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     3301 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattgga tccgtcgacg
     3361 ccaccatggt gtctaagggc gaagagctga tcaaggaaaa tatgcgtatg aaggtggtca
     3421 tggaaggttc ggtcaacggc caccaattca aatgcacagg tgaaggagaa ggcagaccgt
     3481 acgagggagt gcaaaccatg aggatcaaag tcatcgaggg aggacccctg ccatttgcct
     3541 ttgacattct tgccacgtcg ttcatgtatg gcagccgtac ctttatcaag tacccggccg
     3601 acatccctga tttctttaaa cagtcctttc ctgagggttt tacttgggaa agagttacga
     3661 gatacgaaga tggtggagtc gtcaccgtca cgcaggacac cagccttgag gatggcgagc
     3721 tcgtctacaa cgtcaaggtc agaggggtaa actttccctc caatggtccc gtgatgcaga
     3781 agaaaaccaa gggttgggag cctaatacag agatgatgta tccagcagat ggtggtctga
     3841 gaggatacac tgacatcgca ctgaaagttg atggtggtgg ccacctgcac tgcaacttcg
     3901 tgacaactta caggtcaaaa aagaccgtcg ggaacatcaa gatgcccggt gtccatgccg
     3961 ttgatcaccg cctggaaagg atcgaggaga gtgacaatga aacctacgta gtgcaaagag
     4021 aagtggcagt tgccaaatac agcaaccttg gtggtggcat ggacgagctg tacaagtaac
     4081 tcgagataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     4141 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     4201 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttagttc ttgccacggc
     4261 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     4321 caattccgtg gtgtttattt gtgaaatttg tgatgctatt gctttatttg taaccatcta
     4381 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     4441 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gagatgtggg
     4501 aggtttttta aagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg
     4561 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     4621 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt
     4681 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     4741 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     4801 cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     4861 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     4921 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     4981 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     5041 ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga tttataaggg
     5101 attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     5161 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     5221 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     5281 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     5341 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     5401 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     5461 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5521 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5581 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5641 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5701 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5761 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5821 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5881 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5941 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     6001 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     6061 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     6121 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     6181 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     6241 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     6301 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6361 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6421 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6481 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6541 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6601 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6661 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6721 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6781 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6841 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6901 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6961 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     7021 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     7081 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     7141 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     7201 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tc
//
