LOCUS       sequence-266213-        3198 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector for multi-gRNA cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3198)
  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 3198)
  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: pGEP179_pX330K
FEATURES             Location/Qualifiers
     source          1..3198
                     /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        764..839
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(925..1230)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGL"
     primer_bind     1080..1102
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1094..1111
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1095..1111
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1121..1140
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1121..1139
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1187..1207)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1187..1205)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1226..1242)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1226..1242)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1239..1261)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1250..1266
                     /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(1274..1280,1281..1298,1299..1304))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1319..1340
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(1427..1526)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1638..1657)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1649..2458
                     /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(1716..1735)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2548..3136
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3037..3056
                     /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 acaccgggtc ttcgagaaga cctgttttag agctagaaat
      781 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
      841 tttttgtttt agagctagaa atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg
      901 cgccaattct gcagacaaat ggctctagag accggcgccg ctacagggcg cgtcccattc
      961 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
     1021 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
     1081 ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg taatacgact cactataggg
     1141 cgaattgggt accgggcccc ccctcgaggt cctccagctt ttgttccctt tagtgagggt
     1201 taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     1261 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     1321 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccac cggtggtctc
     1381 tctagaggta cccgttacat aacttacggt aaatggcccg atctagaccc agctttcttg
     1441 tacaaagttg gcattataag aaagcattgc ttatcaattt gttgcaacga acaggtcact
     1501 atcagtcaaa ataaaatcat tatttgccat ccagctgcag ctctggcccg tgtctcaaaa
     1561 tctctgatgt tacattgcac aagataaaaa tatatcatca tgaacaataa aactgtctgc
     1621 ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcgaggcc
     1681 gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt
     1741 cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag cccgatgcgc cagagttgtt
     1801 tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa
     1861 ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga
     1921 tgcatggtta ctcaccactg cgatccccgg aaaaacagca ttccaggtat tagaagaata
     1981 tcctgattca ggtgaaaata ttgttgatgc gctggcagtg tccctgcgcc ggttgcattc
     2041 gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca
     2101 atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg
     2161 gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt
     2221 cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg
     2281 ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg
     2341 gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat
     2401 tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaatc
     2461 agaattggtt aattggttgt aacattattc agattgggcc ccgttccact gagcgtcaga
     2521 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     2581 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2641 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     2701 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     2761 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     2821 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     2881 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     2941 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     3001 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3061 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3121 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3181 gccttttgct cacatggt
//
