LOCUS       40924_29421        7477 bp DNA     circular SYN 13-MAY-2021
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)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7477)
  AUTHORS   .
  TITLE     Direct Submission
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     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7477
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        12..238
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             239..419
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    466..591
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    1084..1317
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1502..1546
                     /codon_start=1
                     /label=gp41 peptide
                     /note="antigenic peptide corresponding to amino acids 655
                     to 669 of the HIV envelope protein gp41 (Lutje Hulsik et 
                     al., 2013)"
                     /translation="KNEQELLELDKWASL"
     promoter        1721..2221
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     CDS             2233..2628
                     /codon_start=1
                     /label=BSD
                     /note="blasticidin S deaminase"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    2688..2805
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     promoter        2923..3163
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        3192..3267
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(3279..3303)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3341..3929
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     LTR             4001..4234
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4312..4446
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      4473..4608
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        complement(4629..4647)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4657..4673)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      4815..5270
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        5296..5400
                     /label=AmpR promoter
     CDS             5401..6258
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      6432..7020
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7174..7191
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7308..7329
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        7344..7374
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7382..7398
                     /label=lac operator
                     /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     7406..7422
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        7443..7461
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tgcaagctta atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag
       61 ttagcaacat gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag
      121 gtggtacgat cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac
      181 cactgaattg ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg
      241 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
      301 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
      361 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
      421 tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg
      481 actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca
      541 aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag
      601 cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa
      661 tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct
      721 ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt
      781 cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg
      841 catcaaagga tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa
      901 aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga
      961 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1021 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1081 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1141 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1201 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1261 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1321 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1381 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     1441 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     1501 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     1561 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     1621 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     1681 attatcgttt cagacccacc tcccaacccc gaggggaccc ttgcgccttt tccaaggcag
     1741 ccctgggttt gcgcagggac gcggctgctc tgggcgtggt tccgggaaac gcagcggcgc
     1801 cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc gtcacccgga tcttcgccgc
     1861 tacccttgtg ggccccccgg cgacgcttcc tgctccgccc ctaagtcggg aaggttcctt
     1921 gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca cgtctcacta gtaccctcgc
     1981 agacggacag cgccagggag caatggcagc gcgccgaccg cgatgggctg tggccaatag
     2041 cggctgctca gcagggcgcg ccgagagcag cggccgggaa ggggcggtgc gggaggcggg
     2101 gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg tgttccgcat tctgcaagcc
     2161 tccggagcgc acgtcggcag tcggctccct cgttgaccga atcaccgacc tctctcccca
     2221 gggggtacca ccatggccaa gcctttgtct caagaagaat ccaccctcat tgaaagagca
     2281 acggctacaa tcaacagcat ccccatctct gaagactaca gcgtcgccag cgcagctctc
     2341 tctagcgacg gccgcatctt cactggtgtc aatgtatatc attttactgg gggaccttgt
     2401 gcagaactcg tggtgctggg cactgctgct gctgcggcag ctggcaacct gacttgtatc
     2461 gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct gcggacggtg ccgacaggtg
     2521 cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg acagtgatgg acagccgacg
     2581 gcagttggga ttcgtgaatt gctgccctct ggttatgtgt gggagggcct gcagctgcag
     2641 tagtaagaat tctagatctt gagacaaatg gcagtattca tccacaattt taaaagaaaa
     2701 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata
     2761 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg
     2821 gacagcagag atccactttg gcgccggctc gagtgtacaa aaaagcaggc tttaaaggaa
     2881 ccaattcagt cgactggatc cggtaccaag gtcgggcagg aagagggcct atttcccatg
     2941 attccttcat atttgcatat acgatacaag gctgttagag agataattag aattaatttg
     3001 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
     3061 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
     3121 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac cggggccact
     3181 agggacagga tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
     3241 cttgaaaaag tggcaccgag tcggtgcttt ttttctagac ccagctttct tgtacaaagt
     3301 tggcattagc tagcgctaac cggtggcgcg ttaagtcgac aatcaacctc tggattacaa
     3361 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     3421 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     3481 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     3541 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     3601 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     3661 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     3721 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat
     3781 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     3841 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     3901 tcggatctcc ctttgggccg cctccccgcg tcgactttaa gaccaatgac ttacaaggca
     3961 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc
     4021 caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga
     4081 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     4141 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     4201 agaccctttt agtcagtgtg gaaaatctct agcagtacgt atagtagttc atgtcatctt
     4261 attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag gaacttgttt
     4321 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4381 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     4441 tggctctagc tatcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     4501 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     4561 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta gggacgtacc
     4621 caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg
     4681 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     4741 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     4801 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4861 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     4921 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     4981 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     5041 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     5101 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     5161 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     5221 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     5281 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5341 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5401 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5461 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5521 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5581 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5641 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5701 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5761 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5821 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5881 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5941 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     6001 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     6061 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     6121 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6181 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6241 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6301 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     6361 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6421 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     6481 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6541 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     6601 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6661 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6721 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6781 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6841 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6901 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6961 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     7021 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     7081 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     7141 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     7201 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     7261 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     7321 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     7381 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc
     7441 gcaattaacc ctcactaaag ggaacaaaag ctggagc
//
