LOCUS       sequence-410556-        8727 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A plasmid for expression of SaCas9-sgRNA targeting donor plasmids.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8727)
  AUTHORS   Kim J, Kratz AF, Chen S, Sheng J, Kim HK, Zhang L, Singh BK, Chavez 
            A
  TITLE     High-throughput tagging of endogenous loci for rapid 
            characterization of protein function.
  JOURNAL   Sci Adv. 2024 May 3;10(18):eadg8771. doi: 10.1126/sciadv.adg8771. 
            Epub 2024 May 1.
  PUBMED    38691600
REFERENCE   2  (bases 1 to 8727)
  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: "Sci Adv."; 
            date: "2024-05-3"; volume: "10(18):eadg8771. doi"; pages: " 
            10.1126/sciadv.adg8771. Epub 2024 May 1"
COMMENT     Sequence Label: pDNR-gRNA
FEATURES             Location/Qualifiers
     source          1..8727
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        306..579
                     /label=chicken beta-actin promoter
     intron          580..1596
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1520..1542
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1604..1623
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1630..2007
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MSAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRD
                     DVTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWG
                     REFALRDPAGNCVHFVAEEQD"
     misc_feature    2031..2619
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2084..2104)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2502..2513)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2826..3459
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(3588..3604)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3588..3604)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3601..3623)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3612..3628
                     /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)."
     promoter        complement(join(3636..3642,3643..3660,3661..3666))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3681..3702
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3819..3836)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3990..4578)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4070..4089)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4749..5540,5541..5609))
                     /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     5372..5391
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5610..5714)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    5762..5896
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5799..5818)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5853..5872
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             6064..6697
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    6744..6869
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7366..7599
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7784..7828
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    8091..8206
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     promoter        8309..8557
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8309..8329
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8480..8499
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8579..8654
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc
      361 cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg
      421 gggcgcgcgc caggcggggc ggggsggggg gggggggggg gsggggsgrg gcggagaggt
      481 gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg
      541 cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt
      601 cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg actgaccgcg
      661 ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg
      721 gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg gctccgggag
      781 ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg cgtggggagc
      841 gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt
      901 tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg gcggtgcccc gcggtgcggg
      961 gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg gtgagcaggg
     1021 ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc cccctccccg agttgctgag
     1081 cacggcccgg cttcgggtgc ggggctccgt acggggcgtg gcgcggggct cgccgtgccg
     1141 ggcggggggt ggcggcaggk gggggtgccg ggcggggcgg ggccgcctcg ggccggggrg
     1201 ggctcggggg aggggcgcgg cggcccccgg agcgccggcg gctgtcgagg cgcggcgagc
     1261 cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc tttgtcccaa
     1321 atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc cctctagcgg gcgcggggcg
     1381 aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg gccttcgtgc gtcgccgcgc
     1441 cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc ggggggacgg ctgccttcgg
     1501 gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc tagagcctct
     1561 gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt gctggttatt
     1621 gtggccacca tgagtgccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc
     1681 gccggagcgg tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac
     1741 gacttcgccg gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag
     1801 gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc
     1861 gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag
     1921 atcggcgagc agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg
     1981 cacttcgtgg ccgaggagca ggactgatgt acaagtaaag cgtctggaac aatcaacctc
     2041 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     2101 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     2161 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     2221 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     2281 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     2341 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     2401 acaattccgt ggtgttgtcg gggaagctga cgtcctttcc atggctgctc gcctgtgttg
     2461 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     2521 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     2581 ctcagacgag tcggatctcc ctttgggccg cctccccgcc tggaattaat tctgcagtcg
     2641 agacctagaa aaacatggag caatcacaag tagcaataca gcagctacca atgctgattg
     2701 tgcctggcta gaagcacaag aggaggagga ggtgggtttt ccagtcacac ctcaggtacc
     2761 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaagag
     2821 gggactggaa gggctaattc actcccaacg aagacaagat atccttgatc tgtggatcta
     2881 ccacacacaa ggctacttcc ctgattagca gaactacaca ccagggccag gggtcagata
     2941 tccactgacc tttggatggt gctacaagct agtaccagtt gagccagata aggtagaaga
     3001 ggccaataaa ggagagaaca ccagcttgtt acaccctgtg agcctgcatg ggatggatga
     3061 cccggagaga gaagtgttag agtggaggtt tgacagccgc ctagcatttc atcacgtggc
     3121 ccgagagctg catccggagt acttcaagaa ctgctgatat cgagcttgct acaagggact
     3181 ttccgctggg gactttccag ggaggcgtgg cctgggcggg actggggagt ggcgagccct
     3241 cagatcctgc atataagcag ctgctttttg cctgtactgg gtctctctgg ttagaccaga
     3301 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
     3361 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     3421 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tagtagttca tgtcatctta
     3481 ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg ccttgacatt
     3541 gctagcgttt taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     3601 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     3661 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     3721 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     3781 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     3841 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3901 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3961 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4021 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4081 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4141 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4201 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4261 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4321 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4381 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     4441 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4501 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4561 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4621 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4681 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4741 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4801 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     4861 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     4921 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     4981 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5041 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5101 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5161 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5221 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5281 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5341 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5401 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5461 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5521 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5581 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5641 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5701 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat
     5761 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     5821 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     5881 ttatcatgtc tggatcaact ggataactca agctaaccaa aatcatccca aacttcccac
     5941 cccataccct attaccactg ccaattacct gtggtttcat ttactctaaa cctgtgattc
     6001 ctctgaatta ttttcatttt aaagaaattg tatttgttaa atatgtacta caaacttagt
     6061 agttggaagg gctaattcac tcccaaagaa gacaagatat ccttgatctg tggatctacc
     6121 acacacaagg ctacttccct gattagcaga actacacacc agggccaggg gtcagatatc
     6181 cactgacctt tggatggtgc tacaagctag taccagttga gccagataag gtagaagagg
     6241 ccaataaagg agagaacacc agcttgttac accctgtgag cctgcatggg atggatgacc
     6301 cggagagaga agtgttagag tggaggtttg acagccgcct agcatttcat cacgtggccc
     6361 gagagctgca tccggagtac ttcaagaact gctgatatcg agcttgctac aagggacttt
     6421 ccgctgggga ctttccaggg aggcgtggcc tgggcgggac tggggagtgg cgagccctca
     6481 gatcctgcat ataagcagct gctttttgcc tgtactgggt ctctctggtt agaccagatc
     6541 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
     6601 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
     6661 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
     6721 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
     6781 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
     6841 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
     6901 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
     6961 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
     7021 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
     7081 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
     7141 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
     7201 agcaagcggc cggccgctga tcttcagacc tggaggagga gatatgaggg acaattggag
     7261 aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa
     7321 ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct
     7381 tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca
     7441 ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga
     7501 ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat
     7561 cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg
     7621 aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga
     7681 acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag
     7741 cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt
     7801 attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg
     7861 gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc
     7921 tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca
     7981 cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag gtggagagag
     8041 agacagagac agatccattc gattagtgaa cggatctcga cggtatcgcc tttaaaagaa
     8101 aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca
     8161 tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca
     8221 gggacagcag agatccagtt tatcattagt gaacggatct cgacggtatc gatcacgaga
     8281 ctagcctcga gcggccgccc ccttcaccga gggcctattt cccatgattc cttcatattt
     8341 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa
     8401 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
     8461 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
     8521 cttggcttta tatatcttgt ggaaaggacg aaacaccgaa tcgactcgaa cttcgtgtgt
     8581 tttagtactc tggaaacaga atctactaaa acaaggcaaa atgccgtgtt tatctcgtca
     8641 acttgttggc gagatttttt tgaattctcg acctcgagac aaatggcagt attcgtcatt
     8701 agttcatagc ccatatatgg agttccg
//
