LOCUS       sequence-243621-        6040 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a gRNA together with a Puromycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6040)
  AUTHORS   Heurtier V, Owens N, Gonzalez I, Mueller F, Proux C, Mornico D, 
            Clerc P, Dubois A, Navarro P
  TITLE     The molecular logic of Nanog-induced self-renewal in mouse embryonic
            stem cells.
  JOURNAL   Nat Commun. 2019 Mar 7;10(1):1109. doi: 10.1038/s41467-019-09041-z.
  PUBMED    30846691
REFERENCE   2  (bases 1 to 6040)
  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: "Nat 
            Commun."; date: "2019-03-7"; volume: "10(1):1109. doi"; pages: " 
            10.1038/s41467-019-09041-z"
COMMENT     Sequence Label: PB-gRNA-Puro
FEATURES             Location/Qualifiers
     source          1..6040
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        184..432
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     184..204
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     355..374
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        451..526
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    578..695
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        744..1254
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1173..1191
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1276..1875
                     /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(1276..1295)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1772..1792
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2022..2143
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2033..2052)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2087..2106
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   2674..2708
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(2857..2875)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2896..2912)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2896..2912)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2909..2931)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2920..2936
                     /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(2944..2950,2951..2968,2969..2974))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2989..3010
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3298..3886)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3378..3397)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4057..4848,4849..4917))
                     /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     4680..4699
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4918..5022)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5048..5503)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5185..5206)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5397..5416
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5630..5652
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5644..5661
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5645..5661
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5668..5687
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5668..5686
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   5753..5815
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 catgttgtat tataaaataa gtaattagct taacctatac ataatagaaa caaattatac
       61 ttattagtca gtcagaaaca actttggcac atatcaatat tatgctctcg ttaattaaga
      121 ttagtgaacg gatctcgacg gtatcgatca cgagactagc ctcgagcggc cgcccccttc
      181 accgagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
      241 gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
      301 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
      361 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
      421 ggacgaaaca ccgggtcttc gagaagacct gttttagagc tagaaatagc aagttaaaat
      481 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttaaagaat
      541 tctcgacctc gagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg
      601 gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag
      661 aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag
      721 atccactttg gccgcggctc gagggggttg gggttgcgcc ttttccaagg cagccctggg
      781 tttgcgcagg gacgcggctg ctctgggcgt ggttccggga aacgcagcgg cgccgaccct
      841 gggactcgca cattcttcac gtccgttcgc agcgtcaccc ggatcttcgc cgctaccctt
      901 gtgggccccc cggcgacgct tcctgctccg cccctaagtc gggaaggttc cttgcggttc
      961 gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctca ctagtaccct cgcagacgga
     1021 cagcgccagg gagcaatggc agcgcgccga ccgcgatggg ctgtggccaa tagcggctgc
     1081 tcagcagggc gcgccgagag cagcggccgg gaaggggcgg tgcgggaggc ggggtgtggg
     1141 gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg cattctgcaa gcctccggag
     1201 cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg acctctctcc ccagggggat
     1261 ccaccggagc ttaccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     1321 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     1381 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     1441 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     1501 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     1561 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     1621 caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag
     1681 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
     1741 gtgcccgcct tcctggaaac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
     1801 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     1861 aagcccggtg cctgacgccc gccccacgac ccgcagcgcc cgaccgaaag gagcgcacga
     1921 ccccatgcat cggtaccttt aagaccaatg acttacaagg cagctgtaga tcttagccac
     1981 tttctagagt cggggcggcc ggccgcttcg agcagacatg ataagataca ttgatgagtt
     2041 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     2101 tattgcttta tttgtaacca ttataagctg caataaacaa gttgctcttc cgcttcctcg
     2161 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     2221 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     2281 ggccagcaaa aggccaggaa ccgtaaagct agccaacaag ctcgtcatcg ctttgcagaa
     2341 gagcagagag gatatgctca tcgtctaaag aactacccat tttattatat attagtcacg
     2401 atatctataa caagaaaata tatatataat aagttatcac gtaagtagaa catgaaataa
     2461 caatataatt atcgtatgag ttaaatctta aaagtcacgt aaaagataat catgcgtcat
     2521 tttgactcac gcggtcgtta tagttcaaaa tcagtgacac ttaccgcatt gacaagcacg
     2581 cctcacggga gctccaagcg gcgactgaga tgtcctaaat gcacagcgac ggattcgcgc
     2641 tatttagaaa gagagagcaa tatttcaaga atgcatgcgt caattttacg cagactatct
     2701 ttctagggtt aaaaaagatt tgcgctttac tcgacctaaa ctttaaacac gtcatagaat
     2761 cttcgtttga caaaaaccac attgtggcca agctgtgtga cgcgacgcgc gctaaagaat
     2821 ggcaaaccaa gtcgcgcgag gtaccagctt ttgttccctt tagtgagggt taatttcgag
     2881 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     2941 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     3001 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     3061 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     3121 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     3181 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     3241 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3301 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3361 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     3421 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     3481 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3541 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3601 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3661 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3721 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     3781 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     3841 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     3901 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3961 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4021 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     4081 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     4141 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     4201 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     4261 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     4321 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     4381 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     4441 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     4501 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     4561 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     4621 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     4681 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     4741 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     4801 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     4861 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     4921 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     4981 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     5041 gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     5101 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     5161 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     5221 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     5281 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     5341 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     5401 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     5461 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg
     5521 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     5581 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     5641 cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga attggagctc
     5701 ggtattcacg acagcaggct gaataataaa aaaattagaa actattattt aaccctagaa
     5761 agataatcat attgtgacgt acgttaaaga taatcatgcg taaaattgac gcatgtgttt
     5821 tatcggtctg tatatcgagg tttatttatt aatttgaata gatattaagt tttattatat
     5881 ttacacttac atactaataa taaattcaac aaacaattta tttatgttta tttatttatt
     5941 aaaaaaaaac aaaaactcaa aatttcttct ataaagtaac aaaactttta aacattctct
     6001 cttttacaaa aataaactta ttttgtactt taaaaacagt
//
