LOCUS       Exported                8399 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Cloning backbone for sgRNA, also has a barcode that allows detection
            from scRNA-seq.
ACCESSION   .
VERSION     .
KEYWORDS    Lenti-sgRNA(MS2)-puro-barcode
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8399)
  AUTHORS   Xie S, Duan J, Li B, Zhou P, Hon GC
  TITLE     Multiplexed Engineering and Analysis of Combinatorial Enhancer 
            Activity in Single Cells.
  JOURNAL   Mol Cell. 2017 Apr 20;66(2):285-299.e5. doi: 
            10.1016/j.molcel.2017.03.007. Epub 2017 Apr 13.
  PUBMED    28416141
REFERENCE   2  (bases 1 to 8399)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8399
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     27..44
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    161..182
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        197..227
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    235..251
                     /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     240..262
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     259..275
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     259..275
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     294..314
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        296..314
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        342..568
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             569..749
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    796..921
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1414..1647
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1832..1876
                     /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        2054..2294
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     2054..2074
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2225..2244
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2340..2358
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2410..2428
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_feature    2514..2631
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2770..3948
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3001..3939
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3896..3916
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3961..4560
                     /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(3961..3980)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4457..4477
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4576..5164
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4629..4649)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5047..5058)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5253..5486
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5564..5685
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5601..5620)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5655..5674
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5725..5860
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5787..5806
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(5880..5899)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5881..5899)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5909..5926)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5909..5925)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5918..5940)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6067..6522
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6154..6173)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6364..6385
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6548..6652
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6653..7513
                     /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(6871..6890)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7684..8272
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8173..8192
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
       61 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
      121 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
      181 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
      241 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta
      301 accctcacta aagggaacaa aagctggagc tgcaagctta atgtagtctt atgcaatact
      361 cttgtagtct tgcaacatgg taacgatgag ttagcaacat gccttacaag gagagaaaaa
      421 gcaccgtgca tgccgattgg tggaagtaag gtggtacgat cgtgccttat taggaaggca
      481 acagacgggt ctgacatgga ttggacgaac cactgaattg ccgcattgca gagatattgt
      541 atttaagtgc ctagctcgat acataaacgg gtctctctgg ttagaccaga tctgagcctg
      601 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt
      661 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc
      721 cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa
      781 gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga
      841 ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag
      901 agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa
      961 attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc
     1021 agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga
     1081 caaatactgg gacagctaca accatccctt cagacaggat cagaagaact tagatcatta
     1141 tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag
     1201 gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg
     1261 gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata
     1321 taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag
     1381 agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg
     1441 agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt
     1501 attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca
     1561 tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga
     1621 aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg
     1681 caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa
     1741 tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct taatacactc
     1801 cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga
     1861 taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt
     1921 attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat
     1981 agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc
     2041 gaggggaccc agagagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     2101 ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa
     2161 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     2221 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     2281 cttgtggaaa ggacgaaaca ccagagacgg gataccgtct ctgttttaga gctaggccaa
     2341 catgaggatc acccatgtct gcagggccta gcaagttaaa ataaggctag tccgttatca
     2401 acttggccaa catgaggatc acccatgtct gcagggccaa gtggcaccga gtcggtgctt
     2461 ttttaagctt ggcgtaacta gatcttgaga caaatggcag tattcatcca caattttaaa
     2521 agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca
     2581 gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat
     2641 tacagggaca gcagagatcc actttggcgc cggctcgagg gggcccgggt gcaaagatgg
     2701 ataaagtttt aaacagagag gaatctttgc agctaatgga ccttctaggt cttgaaagga
     2761 gtgggaattg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag
     2821 aagttggggg gaggggtcgg caattgatcc ggtgcctaga gaaggtggcg cggggtaaac
     2881 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     2941 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     3001 gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg
     3061 ccttgaatta cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga
     3121 agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt
     3181 gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt
     3241 ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt
     3301 tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt
     3361 tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg
     3421 ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct
     3481 ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg
     3541 tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca
     3601 aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg
     3661 gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg
     3721 cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt
     3781 tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac
     3841 ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag
     3901 cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgacg tacggccacc
     3961 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     4021 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
     4081 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     4141 atcggcaagg tgtgggtcgc ggacgacggc gccgccgtgg cggtctggac cacgccggag
     4201 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     4261 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     4321 cccgcgtggt tcctggccac cgtcggagtc tcgcccgacc accagggcaa gggtctgggc
     4381 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     4441 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     4501 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga
     4561 acgcgttaag tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     4621 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     4681 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     4741 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     4801 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     4861 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     4921 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc
     4981 tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac
     5041 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     5101 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     5161 ccgctgtaca agcaagggcg aggaattctt taagacaatg acttacaagg cagctgtaga
     5221 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag
     5281 acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga
     5341 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     5401 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     5461 ttagtcagtg tggaaaatct ctagcagtac gtatagtagt tcatgtcatc ttattattca
     5521 gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc
     5581 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     5641 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta
     5701 gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     5761 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     5821 gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta cccaattcgc
     5881 cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg
     5941 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     6001 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     6061 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     6121 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     6181 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     6241 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     6301 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     6361 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     6421 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     6481 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     6541 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     6601 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     6661 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     6721 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     6781 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     6841 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     6901 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     6961 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     7021 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     7081 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     7141 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     7201 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     7261 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     7321 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     7381 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     7441 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     7501 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     7561 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     7621 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     7681 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     7741 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     7801 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     7861 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     7921 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     7981 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     8041 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     8101 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     8161 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     8221 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     8281 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     8341 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccg
//
