LOCUS       sequence-122318-        7133 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6 driven SpCas9 sgRNA expression for TS4 site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7133)
  AUTHORS   Bolukbasi MF, Gupta A, Oikemus S, Derr AG, Garber M, Brodsky MH, Zhu
            LJ, Wolfe SA
  TITLE     DNA-binding-domain fusions enhance the targeting range and precision
            of Cas9.
  JOURNAL   Nat Methods. 2015 Oct 19. doi: 10.1038/nmeth.3624.
  PUBMED    26480473
REFERENCE   2  (bases 1 to 7133)
  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 
            Methods. 2015 Oct 19. doi: 10.1038/nmeth.3624."
COMMENT     Sequence Label: pLKO1-puro-U6-sgRNA-TS4
FEATURES             Location/Qualifiers
     source          1..7133
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     426..444
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             529..1128
                     /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(529..548)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1025..1045
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1256..1489
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1561..1682
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1598..1617)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1652..1671
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1722..1857
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1877..1896)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1878..1896)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1906..1923)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1906..1922)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1915..1937)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2064..2519
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2151..2170)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2361..2382
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2545..2649
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2650..2718,2719..3510)
                     /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(2868..2887)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3681..4269
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4170..4189
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4423..4440
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4557..4578
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4593..4598,4599..4616,4617..4623)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4631..4647
                     /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     4636..4658
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4655..4671
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4655..4671
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4690..4710
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4692..4710
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4738..4964
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             4965..5145
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5192..5317
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5810..6043
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6228..6272
                     /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"
     promoter        6570..6818
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6570..6590
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6741..6760
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        6840..6915
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    6964..7081
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        join(7130..7133,1..507)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
ORIGIN
        1 ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg
       61 cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt
      121 cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct
      181 ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa
      241 gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc
      301 cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc
      361 cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc
      421 gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt
      481 gaccgaatca ccgacctctc tccccagggg gatccaccgg agcttaccat gaccgagtac
      541 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
      601 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
      661 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
      721 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
      781 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
      841 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
      901 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
      961 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     1021 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     1081 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac
     1141 gacccgcagc gcccgaccga aaggagcgca cgaccccatg catcggtacc tttaagacca
     1201 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa
     1261 gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg
     1321 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
     1381 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     1441 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tagtagttca
     1501 tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg
     1561 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     1621 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     1681 tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc
     1741 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     1801 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     1861 ggacgtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt
     1921 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     1981 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     2041 gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2101 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2161 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2221 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2281 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2341 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2401 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2461 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     2521 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2581 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     2641 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     2701 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     2761 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     2821 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     2881 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     2941 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     3001 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     3061 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     3121 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     3181 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     3241 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     3301 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     3361 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     3421 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3481 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     3541 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     3601 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3661 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3721 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3781 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     3841 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3901 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3961 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4021 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4081 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4141 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4201 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4261 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4321 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4381 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4441 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     4501 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     4561 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     4621 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     4681 gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctgca agcttaatgt
     4741 agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag caacatgcct
     4801 tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg
     4861 ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact gaattgccgc
     4921 attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct ctctggttag
     4981 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     5041 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     5101 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag
     5161 ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga
     5221 agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag
     5281 cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag
     5341 atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca
     5401 tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac
     5461 atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga
     5521 agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga
     5581 gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac
     5641 caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg agggacaatt
     5701 ggagaagtga attatataaa tataaagtag taaaaattga accattagga gtagcaccca
     5761 ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt
     5821 tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg
     5881 tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta
     5941 ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa
     6001 gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct
     6061 ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc
     6121 tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca
     6181 caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag
     6241 aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc
     6301 tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt
     6361 ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga
     6421 cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa gaaggtggag
     6481 agagagacag agacagatcc attcgattag tgaacggatc tcgacggtat cgatcacgag
     6541 actagcctcg agcggccgcc cccttcaccg agggcctatt tcccatgatt ccttcatatt
     6601 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
     6661 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     6721 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     6781 tcttggcttt atatatcttg tggaaaggac gaaacaccgg agtccgagca gaagaagaag
     6841 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
     6901 gcaccgagtc ggtgcttttt ttgaattctc gacctcgaga caaatggcag tattcatcca
     6961 caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     7021 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
     7081 tcgggtttat tacagggaca gcagagatcc actttggccg cggctcgagg ggg
//
