LOCUS       Exported                8308 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Human pSico-based U6 vector containing murine U6 promoter and sgRNA 
            targeting endogenous CD71 gene.
ACCESSION   .
VERSION     .
KEYWORDS    pU6-sgCD71-2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8308)
  AUTHORS   Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, 
            Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, 
            Weissman JS, Qi LS
  TITLE     CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in 
            Eukaryotes.
  JOURNAL   Cell. 2013 Jul 9. pii: S0092-8674(13)00826-X. doi: 
            10.1016/j.cell.2013.06.044.
  PUBMED    23849981
REFERENCE   2  (bases 1 to 8308)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8308
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(56..75)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        247..626
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        628..826
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             844..1024
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1071..1196
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1695..1928
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2113..2157
                     /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    2455..2572
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    2825..2858
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     2862..2882
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        2965..3040
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        3165..3468
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3469..3672
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3706..4302
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3706..3725)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4202..4222
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4312..4365
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     regulatory      4372..4381
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             4378..5088
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4523..4541)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4791..4810
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    5108..5141
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    5197..5785
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5250..5270)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5668..5679)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5788..5804)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5789..5805)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6314..6494
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6556..7144)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6636..6655)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7315..8175)
                     /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     7938..7957
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8176..8280)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gcttaagcgg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
       61 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
      121 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
      181 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
      241 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      301 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      361 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      421 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      481 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      541 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      601 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      661 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      721 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      781 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt
      841 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
      901 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
      961 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     1021 agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac
     1081 gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta
     1141 cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta
     1201 ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga
     1261 aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta
     1321 atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat
     1381 cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca accctctatt
     1441 gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag
     1501 agcaaaacaa aagtaagacc accgcacagc aagcggccgg ccgcgctgat cttcagacct
     1561 ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa
     1621 attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
     1681 agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg
     1741 ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag
     1801 cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc
     1861 tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa
     1921 cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg
     1981 aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag
     2041 tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa
     2101 aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg
     2161 aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga
     2221 ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag
     2281 ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc
     2341 gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac
     2401 ggatcggcac tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa
     2461 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
     2521 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta
     2581 ttacagggac agcagagatc cagtttggtt agtaccgggc ccgctctaga gatccgacgc
     2641 gccatctcta ggcccgcgcc ggccccctcg cacagacttg tgggagaagc tcggctactc
     2701 ccctgccccg gttaatttgc atataatatt tcctagtaac tatagaggct taatgtgcga
     2761 taaaagacag ataatctgtt ctttttaata ctagctacat tttacatgat aggcttggat
     2821 ttctataact tcgtatagca tacattatac gaagttataa acagcacaaa aggaaactca
     2881 ccctaactgt aaagtaattg tgtgttttga gactataagt atcccttgga gaaccacctt
     2941 gttgggacgc gctagtgtga gtgcgtttta gagctagaaa tagcaagtta aaataaggct
     3001 agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttctc gagtactagg
     3061 atccattagg cggccgcgtg gataaccgta ttaccgccat gcattagtta ttaatagtaa
     3121 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
     3181 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     3241 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     3301 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
     3361 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
     3421 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
     3481 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
     3541 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
     3601 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     3661 ataagcagag ctggtttagt gaaccgtcag atccgctagc ccaccatgac cgagtacaag
     3721 cccacggtgc gcctcgccac ccgcgacgac gtcccccggg ccgtacgcac cctcgccgcc
     3781 gcgttcgccg actaccccgc cacgcgccac accgtcgacc cggaccgcca catcgagcgg
     3841 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     3901 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     3961 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     4021 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     4081 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     4141 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc
     4201 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     4261 gaaggaccgc gcacctggtg catgacccgc aagcccggtg ccggatcggg agagggcaga
     4321 ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaccggt cgccaccatg
     4381 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
     4441 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     4501 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     4561 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     4621 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     4681 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     4741 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     4801 cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
     4861 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
     4921 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
     4981 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
     5041 gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtaaga attcgtcgag
     5101 ggacctaata acttcgtata gcatacatta tacgaagtta tacatgttta agggttccgg
     5161 ttccactagg tacaattcga tatcaagctt atcgataatc aacctctgga ttacaaaatt
     5221 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     5281 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     5341 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc
     5401 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt
     5461 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc
     5521 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
     5581 ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg
     5641 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc
     5701 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg
     5761 atctcccttt gggccgcctc cccgcatcga taccgtcgac ctcgatcgag acctagaaaa
     5821 acatggagca atcacaagta gcaatacagc agctaccaat gctgattgtg cctggctaga
     5881 agcacaagag gaggaggagg tgggttttcc agtcacacct caggtacctt taagaccaat
     5941 gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg
     6001 gctaattcac tcccaacgaa gacaagatat ccttgatctg tggatctacc acacacaagg
     6061 ctacttccct gattggcaga actacacacc agggccaggg atcagatatc cactgacctt
     6121 tggatggtgc tacaagctag taccagttga gcaagagaag gtagaagaag ccaatgaagg
     6181 agagaacacc cgcttgttac accctgtgag cctgcatggg atggatgacc cggagagaga
     6241 agtattagag tggaggtttg acagccgcct agcatttcat cacatggccc gagagctgca
     6301 tccggactgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     6361 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     6421 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     6481 gaaaatctct agcagcatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     6541 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     6601 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     6661 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     6721 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     6781 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     6841 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     6901 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     6961 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     7021 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     7081 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     7141 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     7201 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     7261 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     7321 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     7381 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     7441 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     7501 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     7561 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     7621 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     7681 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     7741 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     7801 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     7861 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     7921 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     7981 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     8041 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     8101 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     8161 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     8221 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     8281 cacatttccc cgaaaagtgc cacctgac
//
