LOCUS       sequence-271416-        3201 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of gRNA i1 targeting TCF4 for CRISPRi; U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3201)
  AUTHORS   Hazelbaker DZ, Beccard A, Angelini G, Mazzucato P, Messana A, Lam D,
            Eggan K, Barrett LE
  TITLE     A multiplexed gRNA piggyBac transposon system facilitates efficient 
            induction of CRISPRi and CRISPRa in human pluripotent stem cells.
  JOURNAL   Sci Rep. 2020 Jan 20;10(1):635. doi: 10.1038/s41598-020-57500-1.
  PUBMED    31959800
REFERENCE   2  (bases 1 to 3201)
  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 Rep."; 
            date: "2020-01-20"; volume: "10(1):635. doi"; pages: " 
            10.1038/s41598-020-57500-1"
COMMENT     Sequence Label: pPN454
FEATURES             Location/Qualifiers
     source          1..3201
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      103..189
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      281..308
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     316..339
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    358..457
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        497..745
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     497..517
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     668..687
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        767..842
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(928..1233)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGL"
     primer_bind     1083..1105
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1097..1114
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1098..1114
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1124..1143
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1124..1142
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1190..1210)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1190..1208)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1229..1245)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1229..1245)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1242..1264)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1253..1269
                     /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(1277..1283,1284..1301,1302..1307))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1322..1343
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(1430..1529)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1641..1660)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1652..2461
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVSLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1719..1738)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2551..3139
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3040..3059
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgctagca tggatctcgg
       61 ggacgtctaa ctactaagcg agagtaggga actgccaggc atcaaataaa acgaaaggct
      121 cagtcggagg actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
      181 aggacaaatc cgccgggagc ggatttgaac gttgtgaagc aacggcccgg agggtggcgg
      241 gcaggacgcc cgccataaac tgccaggcat caaactaagc agaaggccat cctgacggat
      301 ggcctttttg cgtttctaca aactcttcct gttagttagt tacttaagct cgggccccaa
      361 ataatgattt tattttgact gatagtgacc tgttcgttgc aacaaattga taagcaatgc
      421 ttttttataa tgccaacttt gtacaaaaaa gcaggcttta aaggaaccgc cttttgctgg
      481 ccttttgctc acatgtgagg gcctatttcc catgattcct tcatatttgc atatacgata
      541 caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca
      601 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
      661 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
      721 tatcttgtgg aaaggacgaa acaccgtaaa cttgttccaa gtttaggttt tagagctaga
      781 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
      841 gcttttttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc
      901 gtgcgccaat tctgcagaca aatggctcta gagaccggcg ccgctacagg gcgcgtccca
      961 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     1021 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
     1081 ttcccagtca cgacgttgta aaacgacggc cagtgagcgc gcgtaatacg actcactata
     1141 gggcgaattg ggtaccgggc cccccctcga ggtcctccag cttttgttcc ctttagtgag
     1201 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     1261 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     1321 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc caccggtggt
     1381 ctctctagag gtacccgtta cataacttac ggtaaatggc ccgatctaga cccagctttc
     1441 ttgtacaaag ttggcattat aagaaagcat tgcttatcaa tttgttgcaa cgaacaggtc
     1501 actatcagtc aaaataaaat cattatttgc catccagctg cagctctggc ccgtgtctca
     1561 aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc
     1621 tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcgag
     1681 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa
     1741 tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt
     1801 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact
     1861 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga
     1921 tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg tattagaaga
     1981 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgtccctgc gccggttgca
     2041 ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc
     2101 gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg
     2161 ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc
     2221 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat
     2281 aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct
     2341 atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg
     2401 tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta
     2461 atcagaattg gttaattggt tgtaacatta ttcagattgg gccccgttcc actgagcgtc
     2521 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     2581 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     2641 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     2701 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     2761 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     2821 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     2881 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     2941 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     3001 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     3061 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     3121 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     3181 ctggcctttt gctcacatgg t
//
