LOCUS       sequence-175035-        5967 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For cloning and expression of sgRNA together with Venus expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5967)
  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 5967)
  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: "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..5967
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        39..287
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     39..59
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     210..229
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        306..381
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        416..795
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        797..1074
                     /label=chicken beta-actin promoter
     intron          1075..2086
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2010..2032
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2099..2118
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2099..2117
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2131..2151
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2152..2154,2155..2157,2158..2868)
                     /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(2197..2218)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2458..2477)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2805..2826
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2907..2923)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2907..2923)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2937..2954)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2943..3167
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3216..3233)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3216..3232)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3225..3247)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(3373..3828)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3510..3531)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3722..3741
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3855..3959
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3960..4028,4029..4820)
                     /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(4178..4197)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4991..5579
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5480..5499
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5733..5750
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5867..5888
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5903..5908,5909..5926,5927..5933)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5941..5957
                     /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     join(5946..5967,1)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     join(5965..5967,1..14)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5965..5967,1..14)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gaaacagcta tgaccatgag gcgcgccccc ccttcaccga gggcctattt cccatgattc
       61 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
      121 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
      181 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
      241 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggg tcttcgagaa
      301 gacctgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
      361 aaagtggcac cgagtcggtg ctttttttga tccaagggtg gggcgcgccg gattcgacat
      421 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat
      481 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
      541 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
      601 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
      661 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
      721 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
      781 atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc
      841 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg
      901 gcgggggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg
      961 gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt
     1021 atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc
     1081 gctgcgtcgc gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg
     1141 ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc
     1201 tgtaattagc gcttggttta atgacggctc gtttcttttc tgtggctgcg tgaaagcctt
     1261 aaagggctcc gggagggccc tttgtgcggg ggggagcggc tcggggggtg cgtgcgtgtg
     1321 tgtgtgcgtg gggagcgccg cgtgcggccc gcgctgcccg gcggctgtga gcgctgcggg
     1381 cgcggcgcgg ggctttgtgc gctccgcgtg tgcgcgaggg gagcgcggcc gggggcggtg
     1441 ccccgcggtg cgggggggct gcgaggggaa caaaggctgc gtgcggggtg tgtgcgtggg
     1501 ggggtgagca gggggtgtgg gcgcggcggt cgggctgtaa cccccccctg cacccccctc
     1561 cccgagttgc tgagcacggc ccggcttcgg gtgcggggct ccgtgcgggg cgtggcgcgg
     1621 ggctcgccgt gccgggcggg gggtggcggc aggtgggggt gccgggcggg gcggggccgc
     1681 ctcgggccgg ggagggctcg ggggaggggc gcggcggccc cggagcgccg gcggctgtcg
     1741 aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg cgcagggact
     1801 tcctttgtcc caaatctggc ggagccgaaa tctgggaggc gccgccgcac cccctctagc
     1861 gggcgcgggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag ggccttcgtg
     1921 cgtcgccgcg ccgccgtccc cttctccatc tccagcctcg gggctgccgc agggggacgg
     1981 ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc
     2041 tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagatcc ttaattaata
     2101 atacgactca ctataggggc cgccaccatg cccaagaaga agaggaaggt gatggtgagc
     2161 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     2221 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     2281 accctgaagc tgatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     2341 accctgggct acggcctgca gtgcttcgcc cgctaccccg accacatgaa gcagcacgac
     2401 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     2461 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     2521 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     2581 tacaactaca acagccacaa cgtctatatc accgccgaca agcagaagaa cggcatcaag
     2641 gccaacttca agatccgcca caacatcgag gacggcggcg tgcagctcgc cgaccactac
     2701 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     2761 taccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     2821 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggg cggaggcgga
     2881 ggcggaggca cgcgtaaatg attgcagatc cactagttct agagctcgct gatcagcctc
     2941 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     3001 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     3061 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     3121 ttgggaagag aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     3181 aagaaccagc tgggctcgag gctagagcgg ccgccactgg ccgtcgtttt acaacgtcgt
     3241 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     3301 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     3361 aatggcgaat ggaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     3421 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     3481 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     3541 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     3601 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     3661 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     3721 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     3781 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt
     3841 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     3901 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     3961 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     4021 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     4081 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     4141 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     4201 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     4261 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     4321 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     4381 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     4441 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     4501 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     4561 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     4621 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     4681 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     4741 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     4801 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     4861 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     4921 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     4981 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     5041 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     5101 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     5161 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     5221 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     5281 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     5341 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     5401 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     5461 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     5521 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     5581 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     5641 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     5701 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     5761 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     5821 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     5881 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     5941 ttgtgagcgg ataacaattt cacacag
//
