LOCUS       Exported                8887 bp ds-DNA     circular SYN 30-OCT-2018
DEFINITION  CRISPRi library sequence 8888 bps.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8887)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 30, 2018 from SnapGene 4.2.5
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8887
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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."
     misc_feature    2455..2572
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     misc_feature    2631..2944
                     /label=mU6 Promoter
     protein_bind    2825..2858
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    2942..2944
                     /label=Constant Appended Prefix
     misc_feature    2945..2964
                     /label=sgRNA insert
     misc_feature    2965..3057
                     /label=Modified sgRNA Stem Loop
     misc_feature    2965..2977
                     /label=Constant Appended Suffix
     promoter        3127..3338
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     primer_bind     complement(3180..3199)
                     /label=CRISPRi_PCR_Rev
     intron          3339..4277
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     CDS             4298..4894
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="*PSTSPRCASPPATTSPGPYAPSPPRSPTTPPRATPSTRTATSSG
                     SPSCKNSSSRASGSTSARCGSRTTAPRWRSGPRRRASKRGRCSPRSARAWPS*AVPGWP
                     RSNRWKASWRRTGPRSPRGSWPPSASRPTTRARVWAAPSCSPEWRRPSAPGCPPSWRPP
                     RPATSPSTSGSASPSPPTSRCPKDRAPGA*PASPVP"
     CDS             4904..4957
                     /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="RAEEVC*HAVTSRRILAH"
     CDS             4970..5668
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="AS*LRRTCT*SCTWRAPWTTITSSAHPRAKASPTRAPRP*ESRWS
                     RAALSPSPSTSWLLASSTAARPSSTTPRASPTSSSSPSLRASHGRESPHTKTGAC*PLP
                     RTPASRTAASSTTSRSEG*TSHPTAL*CRRKHSAGRPSPRRCTPLTAAWKAETTWP*SS
                     WAGAI*SQTSRPHIDPRNPLRTSRCLASTMWTTDWKESRRPTTRPTSSSTRWQWPDTAT
                     SLANWGTSLIK"
     protein_bind    5688..5721
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    5777..6365
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             complement(6248..6259)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="D*GP"
     primer_bind     complement(6368..6384)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             6894..7074
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7136..7724)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7895..8755)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="YEYSTFPCRPYSLFCGILPSCFCSPRNAGESKRC*RSVGCTSGLH
                     RTGSQQR*DP*EFSPRRTFSNDEHF*SSAMWRGIIPY*RRARATRSPHTLFSE*LG*VL
                     TSHRKASYGWHDSKRIMQCCHNHE**HCGQLTSDNDRRTEGANRFFAQHGGSCNSP*SL
                     GTGAE*SHTKRRA*HHDACSNGNNVAQTINWRTTYSSFPATINRLDGGG*SCRTTSALG
                     PSGWLVYC**IWSR*AWVSRYHCSTGARW*ALPYRSYLHDGESGNYG*TK*TDR*DRCL
                     TD*ALV"
     promoter        complement(8756..8860)
                     /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 cacggacttg 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 gttggtgcag cactaggcgg tgcatttaag agctaagctg gaaacagcat agcaagttta
     3001 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttctcg
     3061 agtactagga tccattaggc ggccgcgtgg ataaccgtat taccgccatg catgtgcccg
     3121 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
     3181 ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
     3241 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
     3301 cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg
     3361 cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacctggct
     3421 gcagtacgtg attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc
     3481 ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg
     3541 ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct
     3601 agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt
     3661 aaatgcgggc caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg
     3721 ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag
     3781 aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc
     3841 gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga
     3901 aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg
     3961 agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc
     4021 ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgcgctt
     4081 ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac
     4141 tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt
     4201 tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt
     4261 ttttcttcca tttcaggtgt cgtgagctag cccaccatga ccgagtacaa gcccacggtg
     4321 cgcctcgcca cccgcgacga cgtcccccgg gccgtacgca ccctcgccgc cgcgttcgcc
     4381 gactaccccg ccacgcgcca caccgtcgac ccggaccgcc acatcgagcg ggtcaccgag
     4441 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     4501 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     4561 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     4621 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     4681 ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     4741 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     4801 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     4861 cgcacctggt gcatgacccg caagcccggt gccggatcgg gagagggcag aggaagtctg
     4921 ctaacatgcg gtgacgtcga ggagaatcct ggcccaccgg tcgccaccag cgagctgatt
     4981 aaggagaaca tgcacatgaa gctgtacatg gagggcaccg tggacaacca tcacttcaag
     5041 tgcacatccg agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggtg
     5101 gtcgagggcg gccctctccc cttcgccttc gacatcctgg ctactagctt cctctacggc
     5161 agcaagacct tcatcaacca cacccagggc atccccgact tcttcaagca gtccttccct
     5221 gagggcttca catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc
     5281 caggacacca gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac
     5341 ttcacatcca acggccctgt gatgcagaag aaaacactcg gctgggaggc cttcaccgag
     5401 acgctgtacc ccgctgacgg cggcctggaa ggcagaaacg acatggccct gaagctcgtg
     5461 ggcgggagcc atctgatcgc aaacatcaag accacatata gatccaagaa acccgctaag
     5521 aacctcaaga tgcctggcgt ctactatgtg gactacagac tggaaagaat caaggaggcc
     5581 aacaacgaga cctacgtcga gcagcacgag gtggcagtgg ccagatactg cgacctccct
     5641 agcaaactgg ggcacaagct taattaagaa ttcgtcgagg gacctaataa cttcgtatag
     5701 catacattat acgaagttat acatgtttaa gggttccggt tccactaggt acaattcgat
     5761 atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     5821 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     5881 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     5941 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     6001 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     6061 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     6121 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc
     6181 tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac
     6241 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     6301 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     6361 ccgcatcgat accgtcgacc tcgatcgaga cctagaaaaa catggagcaa tcacaagtag
     6421 caatacagca gctaccaatg ctgattgtgc ctggctagaa gcacaagagg aggaggaggt
     6481 gggttttcca gtcacacctc aggtaccttt aagaccaatg acttacaagg cagctgtaga
     6541 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag
     6601 acaagatatc cttgatctgt ggatctacca cacacaaggc tacttccctg attggcagaa
     6661 ctacacacca gggccaggga tcagatatcc actgaccttt ggatggtgct acaagctagt
     6721 accagttgag caagagaagg tagaagaagc caatgaagga gagaacaccc gcttgttaca
     6781 ccctgtgagc ctgcatggga tggatgaccc ggagagagaa gtattagagt ggaggtttga
     6841 cagccgccta gcatttcatc acatggcccg agagctgcat ccggactgta ctgggtctct
     6901 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
     6961 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
     7021 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagcatgtg
     7081 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     7141 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     7201 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     7261 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     7321 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     7381 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     7441 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     7501 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     7561 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     7621 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     7681 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     7741 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     7801 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     7861 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     7921 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     7981 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     8041 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     8101 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     8161 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     8221 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     8281 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     8341 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     8401 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     8461 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     8521 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     8581 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     8641 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     8701 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     8761 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     8821 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     8881 acctgac
//
