LOCUS       sequence-434030-        7824 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty concatmer vector in which 4 gRNAs can be inserted.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7824)
  AUTHORS   Andersson-Rolf A, Merenda A, Mustata RC, Li T, Dietmann S, Koo BK
  TITLE     Simultaneous paralogue knockout using a CRISPR-concatemer in mouse 
            small intestinal organoids.
  JOURNAL   Dev Biol. 2016 Oct 27. pii: S0012-1606(16)30479-1. doi: 
            10.1016/j.ydbio.2016.10.016.
  PUBMED    27983963
REFERENCE   2  (bases 1 to 7824)
  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: "Dev Biol. 
            2016 Oct 27. pii: S0012-1606(16)30479-1. doi: 
            10.1016/j.ydbio.2016.10.016."
COMMENT     Sequence Label: 4 gRNA concatemer
FEATURES             Location/Qualifiers
     source          1..7824
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        11..259
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        276..351
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             358..381
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        404..652
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        671..746
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             752..778
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             752..778
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        802..1050
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1071..1146
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1215..1463
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1479..1553
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1576..2075
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1611..1634)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2008..2027
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2096..2695
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2096..2115)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2592..2612
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2795..3309
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    3471..3487
                     /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(3495..3501,3502..3519,3520..3525))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3540..3561
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3678..3695)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3849..4437)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3929..3948)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4608..5399,5400..5468))
                     /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     5231..5250
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5469..5573)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5641..5659
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5697..5719)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5819..5838
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6032..6054
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6182..6201
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             6401..6917
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    6981..7322
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             7389..7805
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     7735..7757
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     7773..7789
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
ORIGIN
        1 cgggcaggaa gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc
       61 tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
      121 gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
      181 ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
      241 gtggaaagga cgaaacaccg ggtcttcgag aagacgtttt agagctagaa atagcaagtt
      301 aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgac
      361 tacaaggacg acgatgacaa ggatctccaa ggtcgggcag gaagagggcc tatttcccat
      421 gattccttca tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt
      481 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
      541 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
      601 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccggagtctt
      661 cgagaagaca gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
      721 ttgaaaaagt ggcaccgagt cggtgctttt ttacccctac gacgtgcccg actacgccgg
      781 atctccaagg tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata
      841 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
      901 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
      961 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
     1021 ttatatatct tgtggaaagg acgaaacacc ggaagtcttc gagaagacaa gttttagagc
     1081 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
     1141 cggtgctttt ttatggcatc aatgcagaag ctgatctcag aggaggacct gggatctcca
     1201 aggtcgggca ggaagagggc ctatttccca tgattccttc atatttgcat atacgataca
     1261 aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa
     1321 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     1381 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     1441 tcttgtggaa aggacgaaac accggtcttc gagaagactt ttagagctag aaatagcaag
     1501 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg
     1561 gatccgaatt ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt
     1621 tagcagcccc gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca
     1681 catccaccgg taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta
     1741 ctcctcccct agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac
     1801 aaatggaagt agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga
     1861 agcgggtagg cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc
     1921 agaggctggg aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg
     1981 gcgcccgaag gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg
     2041 cgctgttctc ctcttcctca tctccgggcc tttcgacctg cagcccaagc ttaccatgac
     2101 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
     2161 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
     2221 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     2281 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt
     2341 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     2401 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     2461 gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     2521 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     2581 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     2641 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgacgccc
     2701 gccccacgac ccgcagcgcc cgaccgaaag gagcgcacga ccccatgcat cgataaaata
     2761 aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg
     2821 caagctagct taagtaacgc cattttgcaa ggcatggaaa atacataact gagaatagag
     2881 aagttcagat caaggttagg aacagagaga cagcagaata tgggccaaac aggatatctg
     2941 tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtccc
     3001 gccctcagca gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat
     3061 gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt
     3121 ctgctccccg agctcaataa aagagcccac aacccctcac tcggcgcgcc agtcctccga
     3181 tagactgcgt cgcccgggta cccgtgtatc caataaaccc tcttgcagtt gcatccgact
     3241 tgtggtctcg ctgttccttg ggagggtctc ctctgagtga ttgactaccc gtcagcgggg
     3301 gtctttcatg ggtaacagtt tcttgaagtt ggagaacaac attctgaggg taggagtcga
     3361 atattaagta atcctgactc aattagccac tgttttgaat ccacatactc caatactcct
     3421 gaaatagttc attatggaca gcgcagaaag agctggggag aattgtgaaa ttgttatccg
     3481 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     3541 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3601 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3661 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3721 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3781 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3841 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     3901 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3961 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4021 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4081 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4141 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4201 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4261 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     4321 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4381 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4441 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4501 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4561 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4621 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4681 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4741 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     4801 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     4861 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     4921 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     4981 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5041 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5101 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5161 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     5221 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5281 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5341 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5401 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5461 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5521 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5581 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     5641 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc
     5701 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     5761 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg
     5821 gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc
     5881 gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa ctgttgggaa
     5941 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     6001 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcg
     6061 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     6121 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     6181 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     6241 cgatgcgtcc ggcgtagagg cgattagtcc aatttgttaa agacaggata tcagtggtcc
     6301 aggctctagt tttgactcaa caatatcacc agctgaagcc tatagagtac gagccataga
     6361 taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac cccacctgta
     6421 ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaata cataactgag
     6481 aatagagaag ttcagatcaa ggttaggaac agagagacag cagaatatgg gccaaacagg
     6541 atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg tccccagatg
     6601 cggtcccgcc ctcagcagtt tctagagaac catcagatgt ttccagggtg ccccaaggac
     6661 ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg
     6721 cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg gcgcgccagt
     6781 cctccgatag actgcgtcgc ccgggtaccc gtattcccaa taaagcctct tgctgtttgc
     6841 atccgaatcg tggactcgct gatccttggg agggtctcct cagattgatt gactgcccac
     6901 ctcgggggtc tttcatttgg aggttccacc gagatttgga gacccctgcc cagggaccac
     6961 cgaccccccc gccgggaggt aagctggcca gcggtcgttt cgtgtctgtc tctgtctttg
     7021 tgcgtgtttg tgccggcatc taatgtttgc gcctgcgtct gtactagtta gctaactagc
     7081 tctgtatctg gcggacccgt ggtggaactg acgagttctg aacacccggc cgcaaccctg
     7141 ggagacgtcc cagggacttt gggggccgtt tttgtggccc gacctgagga agggagtcga
     7201 tgtggaatcc gaccccgtca ggatatgtgg ttctggtagg agacgagaac ctaaaacagt
     7261 tcccgcctcc gtctgaattt ttgctttcgg tttggaaccg aagccgcgcg tcttgtctgc
     7321 tgcagcgctg cagcatcgtt ctgtgttgtc tctgtctgac tgtgtttctg tatttgtctg
     7381 aaaattaggg ccagactgtt accactccct taagtttgac cttaggtcac tggaaagatg
     7441 tcgagcggat cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct
     7501 gctctgcaga atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag
     7561 acctcatcac ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc
     7621 aggtccccta catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc
     7681 cctttgtaca ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg
     7741 aacctcctcg ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag
     7801 gcgccggaat tagatctcca aggt
//
