LOCUS       Exported                8475 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  An empty sgRNA expression vector for expression of sgRNA for Sp Cas9
            (3rd generation lentiviral vector).
ACCESSION   .
VERSION     .
KEYWORDS    pLenti SpBsmBI sgRNA Puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8475)
  AUTHORS   Pham H, Kearns NA, Maehr R
  TITLE     Transcriptional Regulation with CRISPR/Cas9 Effectors in Mammalian 
            Cells.
  JOURNAL   Methods Mol Biol. 2016;1358:43-57. doi: 10.1007/978-1-4939-3067-8_3.
  PUBMED    26463376
REFERENCE   2  (bases 1 to 8475)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8475
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(20..40)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(20..38)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(59..75)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(59..75)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(72..94)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    83..99
                     /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(107..137)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    152..173
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(290..307)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(461..1049)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(541..560)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1220..2080)
                     /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     1843..1862
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2081..2185)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2211..2666)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2348..2369)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2560..2579
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2793..2815
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2807..2824
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2808..2824
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2834..2853
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2834..2852
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      2873..3008
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2927..2946)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    3048..3169
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3059..3078)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3113..3132
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             3241..3474
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    3561..4149
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             3667..3678
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     4076..4096
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     promoter        complement(4778..5959)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4787..5729
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(4810..4830)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     misc_feature    6164..6280
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6311..6551
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     6311..6331
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6482..6501
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        6579..6654
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(6933..6977)
                     /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"
     misc_feature    7162..7395
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    7888..8013
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             8060..8240
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(8241..8467)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
ORIGIN
        1 gcagctccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt
       61 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
      121 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
      181 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
      241 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
      301 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
      361 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
      421 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
      481 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
      541 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
      601 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
      661 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
      721 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
      781 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
      841 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
      901 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
      961 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     1021 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     1081 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     1141 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     1201 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     1261 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     1321 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     1381 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     1441 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     1501 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     1561 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     1621 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     1681 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     1741 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     1801 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     1861 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     1921 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     1981 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     2041 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     2101 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     2161 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag
     2221 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     2281 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     2341 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     2401 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     2461 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     2521 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     2581 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     2641 gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca actgttggga
     2701 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
     2761 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc
     2821 cagtgagcgc gcgtaatacg actcactata gggcgaattg ggtacgtccc taggcctcca
     2881 aaaaagcctc ctcactactt ctggaatagc tcagaggccg aggcggcctc ggcctctgca
     2941 taaataaaaa aaattagtca gccatggggc ggagaatggg cggaactggg cggagttagg
     3001 ggcgggatgg gcggagttag gggcgggata gctagagcca gacatgataa gatacattga
     3061 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     3121 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagttc ctctcactct
     3181 ctgatattca tttctttgca agttataaat actgaataat aagatgacat gaactactac
     3241 tgctagagat tttccacact gactaaaagg gtctgaggga tctctagtta ccagagtcac
     3301 acaacagacg ggcacacact acttgaagca ctcaaggcaa gctttattga ggcttaagca
     3361 gtgggttccc tagttagcca gagagctccc aggctcagat ctggtctaac cagagagacc
     3421 cagtacaagc aaaaagcaga tcttgtcttc gttgggagtg aattagccct tccagtcccc
     3481 ccttttcttt taaaaagtgg ctaagatcta cagctgcctt gtaagtcatt ggtcttaaag
     3541 gtaccgagct cgaattccag gcggggaggc ggcccaaagg gagatccgac tcgtctgagg
     3601 gcgaaggcga agacgcggaa gaggccgcag agccggcagc aggccgcggg aaggaaggtc
     3661 cgctggattg agggccgaag ggacgtagca gaaggacgtc ccgcgcagaa tccaggtggc
     3721 aacacaggcg agcagccatg gaaaggacgt cagcttcccc gacaacacca cggaattgtc
     3781 agtgcccaac agccgagccc ctgtccagca gcgggcaagg caggcggcga tgagttccgc
     3841 cgtggcaata gggaggggga aagcgaaagt cccggaaagg agctgacagg tggtggcaat
     3901 gccccaacca gtgggggttg cgtcagcaaa cacagtgcac accacgccac gttgcctgac
     3961 aacgggccac aactcctcat aaagagacag caaccaggat ttatacaagg aggagaaaat
     4021 gaaagccata cgggaagcaa tagcatgata caaaggcatt aaagcagcgt atccacatag
     4081 cgtaaaagga gcaacatagt taagaatacc agtcaatctt tcacaaattt tgtaatccag
     4141 aggttgattg tcgagttagg cgccaggctt tcgagtcatg caccaggttc gaggtccctc
     4201 aggcacttcc acatcggcgg tgacggtgaa acccaacctt tcatagaaag gaaggttcct
     4261 aggtgcactg gtctcaagaa atgcgggaac accagctcgt tcagcggcct ccacgccggg
     4321 cagaactacg gcgctgccaa gcccctttcc ctggtgatcg ggggaaacgc ctacggtggc
     4381 caggaaccat gcgggctcct taggtcggtg tggcgcgagc aggccttcca tctgctgttg
     4441 cgcggccagt ctagatccag agagctcggc cattcttggg ccgatctcgg cgaacaccgc
     4501 gccggcttcg acgctctctg gtgtggtcca aacggcgacg gcggctccgt catcggccac
     4561 ccacaccttt ccaatgtcga gtcccaccct tgtcaaaaac aattcctgca gttcagtcac
     4621 cctctcgatg tgccggtcgg gatccacggt atggcgggtt gcggggtaat cggcaaaggc
     4681 agcagccagt gttctcactg ctctggggac atcatcgcga gtcgcgagcc gaacagtagg
     4741 cttgtactcg gtcatggtgg cactagaggg gtagttttca cgacacctga aatggaagaa
     4801 aaaaactttg aaccactgtc tgaggcttga gaatgaacca agatccaaac tcaaaaaggg
     4861 caaattccaa ggagaattac atcaagtgcc aagctggcct aacttcagtc tccacccact
     4921 cagtgtgggg aaactccatc gcataaaacc cctcccccca acctaaagac gacgtactcc
     4981 aaaagctcga gaactaatcg aggtgcctgg acggcgcccg gtactccgtg gagtcacatg
     5041 aagcgacggc tgaggacgga aaggcccttt tcctttgtgt gggtgactca cccgcccgct
     5101 ctcccgagcg ccgcgtcctc cattttgagc tccctgcagc agggccggga agcggccatc
     5161 tttccgctca cgcaactggt gccgaccggg ccagccttgc cgcccagggc ggggcgatac
     5221 acggcggcgc gaggccaggc accagagcag gccggccagc ttgagactac ccccgtccga
     5281 ttctcggtgg ccgcgctcgc aggccccgcc tcgccgaaca tgtgcgctgg gacgcacggg
     5341 ccccgtcgcc gcccgcggcc ccaaaaaccg aaataccagt gtgcagatct tggcccgcat
     5401 ttacaagact atcttgccag aaaaaaagcg tcgcaggata tcatcaaaaa ttttaaatgg
     5461 ctagagactt atcgaaagca gcgagacagg cgcgaaggtg ccaccagatt cgcacgcggc
     5521 ggccccagcg cccaggccag gcctcaactc aagcacgagg cgaaggggct ccttaagcgc
     5581 aaggcctcga actctcccac ccacttccaa cccgaagctc gggatcaaga atcacgtact
     5641 gcagccaggg gcgtggaagt aattcaaggc acgcaagggc cataacccgt aaagaggcca
     5701 ggcccgcggg aaccacacac ggcacttacc tgtgttctgg cggcaaaccc gttgcgaaaa
     5761 agaacgttca cggcgactac tgcacttata tacggttctc ccccaccctc gggaaaaagg
     5821 cggagccagt acacgacatc actttcccag tttaccccgc gccaccttct ctaggcaccg
     5881 gttcaattgc cgacccctcc ccccaacttc tcggggactg tgggcgatgt gcgctctgcc
     5941 cactgacggg caccggagcc tcacgcatgc tcttctccac ctcagtgatg acgagagcgg
     6001 gcgggtgagg gggcgggaac gcagcgatct ctgggttcta cgttagtggg agtttaacga
     6061 cggtccctgg gattccccaa ggcaggggcg agtccttttg tatgaattac tctcagctcc
     6121 ggtcggggcg ggttgggggg ggtggtgacg gggaggcatc gataaaattt tgaattttgt
     6181 aatttgtttt tgtaattctt tagtttgtat gtctgttgct attatgtcta ctattctttc
     6241 ccctgcactg taccccccaa tccccccttt tcttttaaaa gttggatccg gtaccaaggt
     6301 cgggcaggaa gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc
     6361 tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
     6421 gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
     6481 ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
     6541 gtggaaagga cgaaacaccg agacgattaa tgcgtctcgt tttagagcta gaaatagcaa
     6601 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
     6661 tctagtgtta accgataccg tcgagatccg ttcactaatc gaatggatct gtctctgtct
     6721 ctctctccac cttcttcttc tattccttcg ggcctgtcgg gtcccctcgg ggttgggagg
     6781 tgggtctgaa acgataatgg tgaatatccc tgcctaactc tattcactat agaaagtaca
     6841 gcaaaaacta ttcttaaacc taccaagcct cctactatca ttatgaataa ttttatatac
     6901 cacagccaat ttgttatgtt aaaccaattc cacaaacttg cccatttatc taattccaat
     6961 aattcttgtt cattcttttc ttgctggttt tgcgattctt caattaagga gtgtattaag
     7021 cttgtgtaat tgttaatttc tctgtcccac tccatccagg tcgtgtgatt ccaaatctgt
     7081 tccagagatt tattactcca actagcattc caaggcacag cagtggtgca aatgagtttt
     7141 ccagagcaac cccaaatccc caggagctgt tgatccttta ggtatctttc cacagccagg
     7201 attcttgcct ggagctgctt gatgccccag actgtgagtt gcaacagatg ctgttgcgcc
     7261 tcaatagccc tcagcaaatt gttctgctgc tgcactatac cagacaataa ttgtctggcc
     7321 tgtaccgtca gcgtcattga cgctgcgccc atagtgcttc ctgctgctcc caagaaccca
     7381 aggaacaaag ctcctattcc cactgctctt ttttctctct gcaccactct tctctttgcc
     7441 ttggtgggtg ctactcctaa tggttcaatt tttactactt tatatttata taattcactt
     7501 ctccaattgt ccctcatatc tcctcctcca ggtctgaaga tcagcggccg cttgctgtgc
     7561 ggtggtctta cttttgtttt gctcttcctc tatcttgtct aaagcttcct tggtgtcttt
     7621 tatctctatc ctttgatgca cacaatagag ggttgctact gtattatata atgatctaag
     7681 ttcttctgat cctgtctgaa gggatggttg tagctgtccc agtatttgtc tacagccttc
     7741 tgatgtttct aacaggccag gattaactgc gaatcgttct agctccctgc ttgcccatac
     7801 tatatgtttt aatttatatt ttttctttcc ccctggcctt aaccgaattt tttcccatcg
     7861 cgatctaatt ctcccccgct taatactgac gctctcgcac ccatctctct ccttctagcc
     7921 tccgctagtc aaaatttttg gcgtactcac cagtcgccgc ccctcgcctc ttgccgtgcg
     7981 cgcttcagca agccgagtcc tgcgtcgaga gagctcctct ggtttccctt tcgctttcaa
     8041 gtccctgttc gggcgccact gctagagatt ttccacactg actaaaaggg tctgagggat
     8101 ctctagttac cagagtcaca caacagacgg gcacacacta cttgaagcac tcaaggcaag
     8161 ctttattgag gcttaagcag tgggttccct agttagccag agagctccca ggctcagatc
     8221 tggtctaacc agagagaccc gtttatgtat cgagctaggc acttaaatac aatatctctg
     8281 caatgcggca attcagtggt tcgtccaatc catgtcagac ccgtctgttg ccttcctaat
     8341 aaggcacgat cgtaccacct tacttccacc aatcggcatg cacggtgctt tttctctcct
     8401 tgtaaggcat gttgctaact catcgttacc atgttgcaag actacaagag tattgcataa
     8461 gactacatta agctt
//
