LOCUS       Exported                6783 bp ds-DNA     circular SYN 15-JUN-2022
DEFINITION  3rd gen lentiviral backbone for cloning and expression of new shRNA 
            sequences. Uses blasticidin for selection..
ACCESSION   .
VERSION     .
KEYWORDS    pLKO.1-blast
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6783)
  AUTHORS   Bryant DM, Datta A, Rodriguez-Fraticelli AE, Peranen J, 
            Martin-Belmonte F, Mostov KE
  TITLE     A molecular network for de novo generation of the apical surface and
            lumen.
  JOURNAL   Nat Cell Biol. 2010 Oct 3. ():.
  PUBMED    20890297
REFERENCE   2  (bases 1 to 6783)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 15, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6783
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     98..115
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    232..253
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        268..298
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    306..322
                     /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     311..333
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     330..346
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     330..346
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     365..385
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        367..385
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        413..639
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             640..820
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    867..992
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1485..1718
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1903..1947
                     /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        2245..2485
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     2245..2265
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2416..2435
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2556..2673
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2722..3232
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3151..3169
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3244..3642
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             3714..3947
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4019..4140
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4056..4075)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4110..4129
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4180..4315
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4242..4261
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(4335..4354)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4336..4354)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4364..4381)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4364..4380)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4373..4395)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4522..4977
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4609..4628)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4819..4840
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5003..5107
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5108..5968
                     /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(5326..5345)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6139..6727
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6628..6647
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
       61 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
      121 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
      181 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
      241 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
      301 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
      361 gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctgcaagctt aatgtagtct
      421 tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca tgccttacaa
      481 ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga tcgtgcctta
      541 ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt gccgcattgc
      601 agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg gttagaccag
      661 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
      721 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
      781 tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact
      841 tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc
      901 gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag
      961 gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc
     1021 gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag
     1081 tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag
     1141 aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac
     1201 ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa
     1261 aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg
     1321 cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga
     1381 agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag
     1441 gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt
     1501 gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag
     1561 gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag
     1621 gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc
     1681 ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga
     1741 aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa
     1801 cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc
     1861 ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta
     1921 ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg
     1981 tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct
     2041 gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac
     2101 ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga
     2161 gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgatc acgagactag
     2221 cctcgagcgg ccgccccctt caccgagggc ctatttccca tgattccttc atatttgcat
     2281 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
     2341 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
     2401 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
     2461 gctttatata tcttgtggaa aggacgaaac accggtccgc aggtatgcac gcgtgaattc
     2521 tcgacctcga gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg
     2581 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa
     2641 ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat
     2701 ccactttggc cgcggctcga gggggttggg gttgcgcctt ttccaaggca gccctgggtt
     2761 tgcgcaggga cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg ccgaccctgg
     2821 gtctcgcaca ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg ctacccttgt
     2881 gggccccccg gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct tgcggttcgc
     2941 ggcgtgccgg acgtgacaaa cggaagccgc acgtctcact agtaccctcg cagacggaca
     3001 gcgccaggga gcaatggcag cgcgccgacc gcgatgggct gtggccaata gcggctgctc
     3061 agcagggcgc gccgagagca gcggccggga aggggcggtg cgggaggcgg ggtgtggggc
     3121 ggtagtgtgg gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc ctccggagcg
     3181 cacgtcggca gtcggctccc tcgttgaccg aatcaccgac ctctctcccc agggggatcc
     3241 accatggcca agcctttgtc tcaagaagaa tccaccctca ttgaaagagc aacggctaca
     3301 atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct ctctagcgac
     3361 ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg tgcagaactc
     3421 gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc
     3481 ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt gcttctcgat
     3541 ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac ggcagttggg
     3601 attcgtgaat tgctgccctc tggttatgtg tgggagggct aaggtacctt taagaccaat
     3661 gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg
     3721 gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt
     3781 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     3841 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     3901 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta gtagttcatg
     3961 tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggaa
     4021 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     4081 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     4141 tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc
     4201 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     4261 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggg
     4321 acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac
     4381 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     4441 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     4501 gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4561 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     4621 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4681 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     4741 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     4801 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     4861 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     4921 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     4981 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5041 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5101 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     5161 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5221 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     5281 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     5341 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     5401 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     5461 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     5521 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     5581 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     5641 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     5701 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     5761 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     5821 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     5881 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     5941 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     6001 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     6061 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     6121 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6181 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6241 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     6301 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     6361 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     6421 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     6481 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     6541 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     6601 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     6661 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     6721 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     6781 tca
//
