LOCUS       Exported                8893 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Lentiviral vector for expression of Flag tagged CRBN-P2A-Hygro 
            casette from a CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    pLC-Flag-CRBN-P2A-Hygro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8893)
  AUTHORS   Patil A, Manzano M, Gottwein E
  TITLE     Genome-wide CRISPR screens reveal genetic mediators of cereblon 
            modulator toxicity in primary effusion lymphoma.
  JOURNAL   Blood Adv. 2019 Jul 23;3(14):2105-2117. doi: 
            10.1182/bloodadvances.2019031732.
  PUBMED    31300418
REFERENCE   2  (bases 1 to 8893)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8893
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             1..1023
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     protein_bind    1043..1076
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    1132..1720
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1185..1205)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1603..1614)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(1723..1739)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1724..1740)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             2249..2429
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(2491..3079)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2571..2590)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3250..4110)
                     /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     3873..3892
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4111..4215)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4290..4309)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4481..4860
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4862..5060
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             5078..5258
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5305..5430
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5927..6160
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6345..6389
                     /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    6687..6804
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        6929..7232
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7233..7436
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      7475..7484
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             7484..7507
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             8837..8893
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
ORIGIN
        1 aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc
       61 gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta
      121 ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt
      181 tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg
      241 gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa
      301 gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg
      361 atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc
      421 ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac
      481 tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg
      541 atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc
      601 aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg
      661 ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt
      721 atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg
      781 ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc
      841 aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc
      901 gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt
      961 gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa
     1021 taggaattcg tcgagggacc taataacttc gtatagcata cattatacga agttatacat
     1081 gtttaagggt tccggttcca ctaggtacaa ttcgatatca agcttatcga taatcaacct
     1141 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     1201 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     1261 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     1321 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     1381 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     1441 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     1501 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
     1561 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     1621 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     1681 cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg tcgacctcga
     1741 tcgagaccta gaaaaacatg gagcaatcac aagtagcaat acagcagcta ccaatgctga
     1801 ttgtgcctgg ctagaagcac aagaggagga ggaggtgggt tttccagtca cacctcaggt
     1861 acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa
     1921 ggggggactg gaagggctaa ttcactccca acgaagacaa gatatccttg atctgtggat
     1981 ctaccacaca caaggctact tccctgattg gcagaactac acaccagggc cagggatcag
     2041 atatccactg acctttggat ggtgctacaa gctagtacca gttgagcaag agaaggtaga
     2101 agaagccaat gaaggagaga acacccgctt gttacaccct gtgagcctgc atgggatgga
     2161 tgacccggag agagaagtat tagagtggag gtttgacagc cgcctagcat ttcatcacat
     2221 ggcccgagag ctgcatccgg actgtactgg gtctctctgg ttagaccaga tctgagcctg
     2281 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt
     2341 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc
     2401 cttttagtca gtgtggaaaa tctctagcag catgtgagca aaaggccagc aaaaggccag
     2461 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     2521 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     2581 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     2641 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     2701 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     2761 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     2821 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     2881 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     2941 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3001 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     3061 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     3121 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     3181 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     3241 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     3301 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     3361 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     3421 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     3481 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     3541 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     3601 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     3661 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     3721 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     3781 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     3841 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     3901 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     3961 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     4021 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     4081 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     4141 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     4201 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     4261 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4321 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
     4381 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
     4441 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt
     4501 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
     4561 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
     4621 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
     4681 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     4741 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     4801 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     4861 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
     4921 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
     4981 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
     5041 tgggaggtct atataagcag cgcgttttgc ctgtactggg tctctctggt tagaccagat
     5101 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     5161 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     5221 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg
     5281 aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc
     5341 acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc
     5401 tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga
     5461 tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta
     5521 tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa
     5581 ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt
     5641 agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa
     5701 gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca
     5761 cagcaagcgg ccggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
     5821 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     5881 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     5941 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     6001 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     6061 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     6121 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     6181 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     6241 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     6301 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     6361 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     6421 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     6481 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     6541 acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga
     6601 gagacagaga cagatccatt cgattagtga acggatcggc actgcgtgcg ccaattctgc
     6661 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt
     6721 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     6781 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttgg
     6841 ttagtaccgg gcccgagagt taattaatta gttattaata gtaatcaatt acggggtcat
     6901 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     6961 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     7021 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     7081 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
     7141 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
     7201 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
     7261 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     7321 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
     7381 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctggtt
     7441 tagtgaaccg tcagatccgc tagcgctacc ggtcgccacc atggattaca aagacgatga
     7501 cgataaggcc ggcgaaggag atcagcagga cgctgcgcac aacatgggca accacctgcc
     7561 gctcctgcct gagagtgagg aagaagatga aatggaagtt gaagaccagg atagtaaaga
     7621 agccaaaaaa ccaaacatca taaattttga caccagtctg ccgacatcac atacatacct
     7681 aggtgctgat atggaagaat ttcatggcag gactttgcac gatgacgaca gctgtcaggt
     7741 gattccagtt cttccacaag tgatgatgat cctgattccc ggacagacat tacctcttca
     7801 gctttttcac cctcaagaag tcagtatggt gcggaattta attcagaaag atagaacctt
     7861 tgctgttctt gcatacagca atgtacagga aagggaagca cagtttggaa caacagcaga
     7921 gatatatgcc tatcgagaag aacaggattt tggaattgag atagtgaaag tgaaagcaat
     7981 tggaagacaa aggttcaaag tccttgagct aagaacacag tcagatggaa tccagcaagc
     8041 taaagtgcaa attcttcccg aatgtgtgtt gccttcaacc atgtctgcag ttcaattaga
     8101 atccctcaat aagtgccaga tatttccttc aaaacctgtc tcaagagaag accaatgttc
     8161 atataaatgg tggcagaaat accagaagag aaagtttcat tgtgcaaatc taacttcatg
     8221 gcctcgctgg ctgtattcct tatatgatgc tgagacctta atggacagaa tcaagaaaca
     8281 gctacgtgaa tgggatgaaa atctaaaaga tgattctctt ccttcaaatc caatagattt
     8341 ttcttacaga gtagctgctt gtcttcctat tgatgatgta ttgagaattc agctccttaa
     8401 aattggcagt gctatccagc gacttcgctg tgaattagac attatgaata aatgtacttc
     8461 cctttgctgt aaacaatgtc aagaaacaga aataacaacc aaaaatgaaa tattcagttt
     8521 atccttatgt gggccgatgg cagcttatgt gaatcctcat ggatatgtgc atgagacact
     8581 tactgtgtat aaggcttgca acttgaatct gataggccgg ccttctacag aacacagctg
     8641 gtttcctggg tatgcctgga ctgttgccca gtgtaagatc tgtgcaagcc atattggatg
     8701 gaagtttacg gccaccaaaa aagacatgtc acctcaaaaa ttttggggct taacgcgatc
     8761 tgctctgttg cccacgatcc cagacactga agatgaaata agtccagaca aagtaatact
     8821 ttgcttggga tccggcgcaa caaacttctc tctgctgaaa caagccggag atgtcgaaga
     8881 gaatcctgga ccg
//
