LOCUS       sequence-175034-        4730 bp    DNA     linear   UNA 14-APR-2025
DEFINITION  For cloning and expression of sgRNA together with Venus expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4730)
  AUTHORS   Yumlu S, Bashir S, Stumm J, Kuhn R
  TITLE     Efficient Gene Editing of Human Induced Pluripotent Stem Cells Using
            CRISPR/Cas9.
  JOURNAL   Methods Mol Biol. 2019;1961:137-151. doi: 
            10.1007/978-1-4939-9170-9_10.
  PUBMED    30912045
REFERENCE   2  (bases 1 to 4730)
  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: "Methods Mol
            Biol."; date: "2019"; volume: "1961:137-151. doi"; pages: " 
            10.1007/978-1-4939-9170-9_10"
COMMENT     Sequence Label: U6-(BbsI)sgRNA_CAG-Venus-bpA
FEATURES             Location/Qualifiers
     source          1..4730
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     3..22
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3..21
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             35..55
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(56..58,59..61,62..772)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(101..122)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(362..381)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     709..730
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(811..827)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(811..827)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(841..858)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    847..1071
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1120..1137)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1120..1136)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1129..1151)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(1277..1732)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1414..1435)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1626..1645
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1759..1863
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1864..1932,1933..2724)
                     /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(2082..2101)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2895..3483
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3384..3403
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3637..3654
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3771..3792
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3807..3812,3813..3830,3831..3837)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3845..3861
                     /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)."
     primer_bind     3850..3872
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3869..3885
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3869..3885
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3910..4158
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3910..3930
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4081..4100
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4177..4252
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        4287..4666
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 aataatacga ctcactatag gggccgccac catgcccaag aagaagagga aggtgatggt
       61 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
      121 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
      181 gctgaccctg aagctgatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
      241 gaccaccctg ggctacggcc tgcagtgctt cgcccgctac cccgaccaca tgaagcagca
      301 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
      361 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
      421 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
      481 ggagtacaac tacaacagcc acaacgtcta tatcaccgcc gacaagcaga agaacggcat
      541 caaggccaac ttcaagatcc gccacaacat cgaggacggc ggcgtgcagc tcgccgacca
      601 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
      661 gagctaccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
      721 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agggcggagg
      781 cggaggcgga ggcacgcgta aatgattgca gatccactag ttctagagct cgctgatcag
      841 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
      901 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
      961 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     1021 aggattggga agagaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     1081 cggaaagaac cagctgggct cgaggctaga gcggccgcca ctggccgtcg ttttacaacg
     1141 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     1201 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     1261 cctgaatggc gaatggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     1321 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     1381 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     1441 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     1501 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     1561 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     1621 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     1681 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc
     1741 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     1801 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     1861 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     1921 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     1981 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     2041 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     2101 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     2161 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     2221 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     2281 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     2341 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     2401 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     2461 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     2521 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     2581 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     2641 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     2701 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     2761 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     2821 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     2881 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     2941 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     3001 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     3061 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3121 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3181 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3241 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3301 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3361 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3421 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     3481 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     3541 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     3601 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     3661 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     3721 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     3781 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     3841 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ggcgcgcccc
     3901 cccttcaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     3961 gttagagaga taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg
     4021 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     4081 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     4141 tggaaaggac gaaacaccgg gtcttcgaga agacctgttt tagagctaga aatagcaagt
     4201 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg
     4261 atccaagggt ggggcgcgcc ggattcgaca ttgattattg actagttatt aatagtaatc
     4321 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     4381 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     4441 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     4501 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     4561 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     4621 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
     4681 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt
//
