LOCUS       sequence-75816-e        7477 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression of AAVS1-targeting sgRNA; insert can be 
            replaced with custom sgRNA (see protocol).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7477)
  AUTHORS   Wang T, Wei JJ, Sabatini DM, Lander ES
  TITLE     Genetic screens in human cells using the CRISPR-Cas9 system.
  JOURNAL   Science. 2014 Jan 3;343(6166):80-4. doi: 10.1126/science.1246981. 
            Epub 2013 Dec 12.
  PUBMED    24336569
REFERENCE   2  (bases 1 to 7477)
  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: 
            "10.1126/science.1246981"; journalName: "Science"; date: "2014-01-3-
            3"; volume: "343"; issue: "6166"; pages: "80-4"
COMMENT     Sequence Label: pLX-sgRNA
FEATURES             Location/Qualifiers
     source          1..7477
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..231
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             232..412
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    459..584
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1077..1310
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1495..1539
                     /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        1714..2214
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2133..2151
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2226..2621
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    2681..2798
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2916..3164
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2916..2936
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3087..3106
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3185..3260
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(3272..3296)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3334..3922
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3387..3407)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3805..3816)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3994..4227
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4305..4426
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4342..4361)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4396..4415
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4466..4601
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4621..4640)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4622..4640)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4650..4667)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4650..4666)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4659..4681)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4808..5263
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4895..4914)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5105..5126
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5289..5393
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5394..5462,5463..6254)
                     /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(5612..5631)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6425..7013
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6914..6933
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7167..7184
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7301..7322
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7337..7342,7343..7360,7361..7367)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7375..7391
                     /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     7380..7402
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7399..7415
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7399..7415
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7434..7454
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7436..7454
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
       61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
      661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
      721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
      781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
      841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
      901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
      961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1321 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1381 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1441 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     1501 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     1561 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     1621 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     1681 tttcagaccc acctcccaac cccgagggga cccttgcgcc ttttccaagg cagccctggg
     1741 tttgcgcagg gacgcggctg ctctgggcgt ggttccggga aacgcagcgg cgccgaccct
     1801 gggtctcgca cattcttcac gtccgttcgc agcgtcaccc ggatcttcgc cgctaccctt
     1861 gtgggccccc cggcgacgct tcctgctccg cccctaagtc gggaaggttc cttgcggttc
     1921 gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctca ctagtaccct cgcagacgga
     1981 cagcgccagg gagcaatggc agcgcgccga ccgcgatggg ctgtggccaa tagcggctgc
     2041 tcagcagggc gcgccgagag cagcggccgg gaaggggcgg tgcgggaggc ggggtgtggg
     2101 gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg cattctgcaa gcctccggag
     2161 cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg acctctctcc ccagggggta
     2221 ccaccatggc caagcctttg tctcaagaag aatccaccct cattgaaaga gcaacggcta
     2281 caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct ctctctagcg
     2341 acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct tgtgcagaac
     2401 tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga
     2461 tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag gtgcttctcg
     2521 atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg acggcagttg
     2581 ggattcgtga attgctgccc tctggttatg tgtgggaggg cctgcagctg cagtagtaag
     2641 aattctagat cttgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
     2701 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
     2761 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
     2821 gagatccact ttggcgccgg ctcgagtgta caaaaaagca ggctttaaag gaaccaattc
     2881 agtcgactgg atccggtacc aaggtcgggc aggaagaggg cctatttccc atgattcctt
     2941 catatttgca tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa
     3001 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     3061 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     3121 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggggcc actagggaca
     3181 ggatgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
     3241 aagtggcacc gagtcggtgc tttttttcta gacccagctt tcttgtacaa agttggcatt
     3301 agctagcgct aaccggtggc gcgttaagtc gacaatcaac ctctggatta caaaatttgt
     3361 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     3421 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     3481 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     3541 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     3601 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     3661 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     3721 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc
     3781 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     3841 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
     3901 tccctttggg ccgcctcccc gcgtcgactt taagaccaat gacttacaag gcagctgtag
     3961 atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa
     4021 gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg
     4081 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
     4141 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
     4201 tttagtcagt gtggaaaatc tctagcagta cgtatagtag ttcatgtcat cttattattc
     4261 agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag
     4321 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4381 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct
     4441 agctatcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     4501 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     4561 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg
     4621 ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg
     4681 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     4741 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     4801 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4861 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     4921 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4981 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     5041 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     5101 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     5161 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     5221 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     5281 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     5341 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     5401 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     5461 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     5521 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     5581 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     5641 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     5701 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     5761 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     5821 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     5881 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     5941 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     6001 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6061 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     6121 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     6181 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     6241 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     6301 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     6361 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     6421 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6481 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6541 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     6601 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6661 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6721 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6781 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6841 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     6901 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     6961 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     7021 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     7081 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     7141 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     7201 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     7261 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     7321 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     7381 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta
     7441 accctcacta aagggaacaa aagctggagc tgcaagc
//
