LOCUS       sequence-238505-        7922 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6 driven sgRNA cloning vector where guide sequences are inserted 
            between BfuAI sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7922)
  AUTHORS   Kearns NA, Genga RM, Enuameh MS, Garber M, Wolfe SA, Maehr R
  TITLE     Cas9 effector-mediated regulation of transcription and 
            differentiation in human pluripotent stem cells.
  JOURNAL   Development. 2014 Jan;141(1):219-23. doi: 10.1242/dev.103341.
  PUBMED    24346702
REFERENCE   2  (bases 1 to 7922)
  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"; doi: "10.1242/dev"; 
            journalName: "Development"; date: "2014-01"; volume: "141"; issue: 
            "1"; pages: "219-23"
COMMENT     Sequence Label: pLKO.1-puro U6 sgRNA BfuAI stuffer
FEATURES             Location/Qualifiers
     source          1..7922
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        129..377
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     129..149
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     300..319
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        411..486
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    535..652
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        701..1211
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1130..1148
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1233..1832
                     /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(1233..1252)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1729..1749
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1960..2193
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2265..2386
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2302..2321)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2356..2375
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2426..2561
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2581..2600)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2582..2600)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2610..2627)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2610..2626)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2619..2641)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2768..3223
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2855..2874)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3065..3086
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3249..3353
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3354..3422,3423..4214)
                     /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(3572..3591)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4385..4973
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4874..4893
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5127..5144
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5261..5282
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     mobile_element  5307..6074
                     /mobile_element_type="insertion sequence:IS1"
                     /label=IS1
                     /note="prokaryotic transposable element"
     promoter        join(6075..6079,6080..6097,6098..6104)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6112..6128
                     /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     6117..6139
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6136..6152
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6136..6152
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6171..6191
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6173..6191
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6219..6445
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6446..6626
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6673..6798
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7291..7524
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7709..7753
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
ORIGIN
        1 gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca
       61 ttcgattagt gaacggatct cgacggtatc gatcacgaga ctagcctcga gcggccgccc
      121 ccttcaccga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
      181 ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt
      241 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
      301 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
      361 ggaaaggacg aaacaccggt gtgcaggtga gtgatcaaga acctgctgac gttttagagc
      421 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
      481 cggtgctttt tttgaattct cgacctcgag acaaatggca gtattcatcc acaattttaa
      541 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
      601 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta
      661 ttacagggac agcagagatc cactttggcc gcggctcgag ggggttgggg ttgcgccttt
      721 tccaaggcag ccctgggttt gcgcagggac gcggctgctc tgggcgtggt tccgggaaac
      781 gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc gtcacccgga
      841 tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc tgctccgccc ctaagtcggg
      901 aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca cgtctcacta
      961 gtaccctcgc agacggacag cgccagggag caatggcagc gcgccgaccg cgatgggctg
     1021 tggccaatag cggctgctca gcagggcgcg ccgagagcag cggccgggaa ggggcggtgc
     1081 gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg tgttccgcat
     1141 tctgcaagcc tccggagcgc acgtcggcag tcggctccct cgttgaccga atcaccgacc
     1201 tctctcccca gggggatcca ccggagctta ccatgaccga gtacaagccc acggtgcgcc
     1261 tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact
     1321 accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc
     1381 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     1441 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     1501 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     1561 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     1621 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     1681 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     1741 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     1801 cctggtgcat gacccgcaag cccggtgcct gacgcccgcc ccacgacccg cagcgcccga
     1861 ccgaaaggag cgcacgaccc catgcatcgg tacctttaag accaatgact tacaaggcag
     1921 ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc
     1981 aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag
     2041 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     2101 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     2161 gaccctttta gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc
     2221 agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag
     2281 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     2341 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct
     2401 agctatcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     2461 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     2521 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg
     2581 ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg
     2641 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     2701 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     2761 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2821 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     2881 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     2941 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     3001 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     3061 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     3121 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     3181 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     3241 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     3301 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     3361 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     3421 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     3481 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     3541 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     3601 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     3661 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     3721 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     3781 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     3841 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     3901 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     3961 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     4021 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     4081 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     4141 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     4201 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     4261 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     4321 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     4381 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     4441 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     4501 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     4561 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     4621 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     4681 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     4741 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     4801 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     4861 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     4921 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     4981 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     5041 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     5101 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     5161 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     5221 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     5281 attaggcacc ccaggcttta cactttggta atgactccaa cttattgata gtgttttatg
     5341 ttcagataat gcccgatgac tttgtcatgc agctccaccg attttgagaa cgacagcgac
     5401 ttccgtccca gccgtgccag gtgctgcctc agattcaggt tatgccgctc aattcgctgc
     5461 gtatatcgct tgctgattac gtgcagcttt cccttcaggc gggattcata cagcggccag
     5521 ccatccgtca tccatatcac cacgtcaaag ggtgacagca ggctcataag acgccccagc
     5581 gtcgccatag tgcgttcacc gaatacgtgc gcaacaaccg tcttccggag actgtcatac
     5641 gcgtaaaaca gccagcgctg gcgcgattta gccccgacat agccccactg ttcgtccatt
     5701 tccgcgcaga cgatgacgtc actgcccggc tgtatgcgcg aggttaccga ctgcggcctg
     5761 agttttttaa gtgacgtaaa atcgtgttga ggccaacgcc cataatgcgg gctgttgccc
     5821 ggcatccaac gccattcatg gccatatcaa tgattttctg gtgcgtaccg ggttgagaag
     5881 cggtgtaagt gaactgcagt tgccatgttt tacggcagtg agagcagaga tagcgctgat
     5941 gtccggcggt gcttttgccg ttacgcacca ccccgtcagt agctgaacag gagggacagc
     6001 tgatagaaac agaagccact ggagcacctc aaaaacacca tcatacacta aatcagtaag
     6061 ttggcagcat caccttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     6121 gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc
     6181 ctcactaaag ggaacaaaag ctggagctgc aagcttaatg tagtcttatg caatactctt
     6241 gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc ttacaaggag agaaaaagca
     6301 ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt gccttattag gaaggcaaca
     6361 gacgggtctg acatggattg gacgaaccac tgaattgccg cattgcagag atattgtatt
     6421 taagtgccta gctcgataca taaacgggtc tctctggtta gaccagatct gagcctggga
     6481 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     6541 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     6601 ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg
     6661 aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc
     6721 gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga
     6781 gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt
     6841 cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg
     6901 gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa
     6961 atactgggac agctacaacc atcccttcag acaggatcag aagaacttag atcattatat
     7021 aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa
     7081 gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc
     7141 gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg aattatataa
     7201 atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt
     7261 ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc
     7321 agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt
     7381 gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct
     7441 gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag
     7501 atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac
     7561 cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca
     7621 cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt
     7681 aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa
     7741 atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt
     7801 cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt
     7861 gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag
     7921 gg
//
