pLH-sgRNA1-boxB-MS2-PP7

LOCUS       pLH-sgRNA1-boxB-MS2-PP7                8337 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  sgRNA1-boxB-MS2-PP7.
ACCESSION   .
VERSION     .
KEYWORDS    pLH-sgRNA1-boxB-MS2-PP7
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8337)
  AUTHORS   Ma H, Tu LC, Naseri A, Huisman M, Zhang S, Grunwald D, Pederson T
  TITLE     Multiplexed labeling of genomic loci with dCas9 and engineered 
            sgRNAs using CRISPRainbow.
  JOURNAL   Nat Biotechnol. 2016 Apr 18. doi: 10.1038/nbt.3526.
  PUBMED    27088723
REFERENCE   2  (bases 1 to 8337)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8337
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     94..111
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    228..249
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        264..294
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    302..318
                     /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     307..329
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     326..342
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     326..342
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     361..381
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        363..381
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        409..635
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             636..816
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    863..988
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1481..1714
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     promoter        2241..2481
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     2241..2261
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2412..2431
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             2830..3135
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     3014..3033
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_RNA        3228..3246
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_feature    3350..3467
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3516..4026
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3945..3963
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4116..5141
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(5165..5181)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5166..5182)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5264..5497
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5569..5690
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5606..5625)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5660..5679
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5730..5865
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5792..5811
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(5885..5904)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5886..5904)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5914..5931)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5914..5930)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5923..5945)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6072..6527
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6159..6178)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6369..6390
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6553..6657
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6658..7518
                     /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(6876..6895)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7689..8277
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8178..8197
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
       61 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      121 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
      181 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
      241 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
      301 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc
      361 gcaattaacc ctcactaaag ggaacaaaag ctggagctgc aagcttaatg tagtcttatg
      421 caatactctt gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc ttacaaggag
      481 agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt gccttattag
      541 gaaggcaaca gacgggtctg acatggattg gacgaaccac tgaattgccg cattgcagag
      601 atattgtatt taagtgccta gctcgataca taaacgggtc tctctggtta gaccagatct
      661 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
      721 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
      781 tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa
      841 agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac
      901 ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta
      961 gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg
     1021 ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg
     1081 ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg
     1141 ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag
     1201 atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga
     1261 caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca
     1321 gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg
     1381 aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa
     1441 agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt
     1501 tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca
     1561 gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc
     1621 aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg
     1681 ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac
     1741 tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga
     1801 tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa
     1861 tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg
     1921 aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata
     1981 taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac
     2041 tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc
     2101 caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca
     2161 gagacagatc cattcgatta gtgaacggat ctcgacggta tcgatcacga gactagcctc
     2221 gagcggccgc ccccttcacc gagggcctat ttcccatgat tccttcatat ttgcatatac
     2281 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
     2341 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     2401 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     2461 tatatatctt gtggaaagga cgaaacaccg gtgtcttcga ggcttactaa aagccagata
     2521 acagtatgcg tatttgcgcg ctgatttttg cggtataaga atatatactg atatgtatac
     2581 ccgaagtatg tcaaaaagag gtgtgctatg aagcagcgta ttacagtgac agttgacagc
     2641 gacagctatc agttgctcaa ggcatatatg atgtcaatat ctccggtctg gtaagcacaa
     2701 ccatgcagaa tgaagcccgt cgtctgcgtg ccgaacgctg gaaagcggaa aatcaggaag
     2761 ggatggctga ggtcgcccgg tttattgaaa tgaacggctc ttttgctgac gagaacaggg
     2821 actggtgaaa tgcagtttaa ggtttacacc tataaaagag agagccgtta tcgtctgttt
     2881 gtggatgtac agagtgatat tattgacacg cccgggcgac ggatggtgat ccccctggcc
     2941 agtgcacgtc tgctgtcaga taaagtctcc cgtgaacttt acccggtggt gcatatcggg
     3001 gatgaaagct ggcgcatgat gaccaccgat atggccagtg tgccggtctc cgttatcggg
     3061 gaagaagtgg ctgatctcag ccaccgcgaa aatgacatca aaaacgccat taacctgatg
     3121 ttctggggaa tataaaccct gcaggacgaa gacccgtttg agagctaggg ccctgaagaa
     3181 gggccctagc aagttcaaat aaggctagtc cgttatcaac ttggccaaca tgaggatcac
     3241 ccatgtctgc agggccaagt ggcaccgagt cggtgcggag cagacgatat ggcgtcgctc
     3301 ctttttttga attctcgacc tcgagacaaa tggcagtatt catccacaat tttaaaagaa
     3361 aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca
     3421 tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca
     3481 gggacagcag agatccactt tggccgcggc tcgagggggt tggggttgcg ccttttccaa
     3541 ggcagccctg ggtttgcgca gggacgcggc tgctctgggc gtggttccgg gaaacgcagc
     3601 ggcgccgacc ctgggtctcg cacattcttc acgtccgttc gcagcgtcac ccggatcttc
     3661 gccgctaccc ttgtgggccc cccggcgacg cttcctgctc cgcccctaag tcgggaaggt
     3721 tccttgcggt tcgcggcgtg ccggacgtga caaacggaag ccgcacgtct cactagtacc
     3781 ctcgcagacg gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc
     3841 aatagcggct gctcagcagg gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag
     3901 gcggggtgtg gggcggtagt gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc
     3961 aagcctccgg agcgcacgtc ggcagtcggc tccctcgttg accgaatcac cgacctctct
     4021 ccccaggggg atccaccgga tcaagcttgc cagctggggc gccctctggt aaggttggga
     4081 agccctgcag atctgatccc ggggggcaat gagatatgaa aaagcctgaa ctcaccgcga
     4141 cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct
     4201 cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc
     4261 gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat
     4321 cggccgcgct cccgattccg gaagtgcttg acattgggga atttagcgag agcctgacct
     4381 attgcatctc ccgccgtgca cagggtgtca cgttgcaaga tctgcctgaa accgaactgc
     4441 ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc
     4501 agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg
     4561 atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca
     4621 ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc
     4681 ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg
     4741 gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg
     4801 tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact
     4861 tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca
     4921 ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg
     4981 cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa
     5041 tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg
     5101 gaaaccgacg ccccagcact cgtccgaggg caaaggaata gagtagatgc cgaccgggat
     5161 ctatcgatac cgtcgacctc gagggggggc ccggtacctt taagaccaat gacttacaag
     5221 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac
     5281 tcccaacgaa gaaaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc
     5341 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
     5401 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
     5461 ctcagaccct tttagtcagt gtggaaaatc tctagcagta gtagttcatg tcatcttatt
     5521 attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggaa cttgtttatt
     5581 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     5641 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     5701 ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     5761 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     5821 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggg acgtacccaa
     5881 ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga
     5941 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     6001 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     6061 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     6121 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     6181 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     6241 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     6301 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     6361 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     6421 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     6481 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     6541 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     6601 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     6661 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     6721 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     6781 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     6841 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     6901 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     6961 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     7021 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     7081 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     7141 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     7201 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     7261 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     7321 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     7381 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     7441 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     7501 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     7561 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     7621 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     7681 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     7741 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     7801 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     7861 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     7921 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     7981 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     8041 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     8101 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     8161 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     8221 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     8281 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatg
//