pLH-sgRNA1-2XPP7

LOCUS       pLH-sgRNA1-2XPP7               8313 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  sgRNA1-2XPP7.
ACCESSION   .
VERSION     .
KEYWORDS    pLH-sgRNA1-2XPP7
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8313)
  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 8313)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8313
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        230..470
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     230..250
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     401..420
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             819..1124
                     /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     1003..1022
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_feature    1315..1432
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1481..1991
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1910..1928
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2081..3106
                     /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(3130..3146)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3131..3147)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             3229..3462
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3534..3655
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3571..3590)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3625..3644
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3695..3830
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3757..3776
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(3850..3869)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3851..3869)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3879..3896)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3879..3895)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3888..3910)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4037..4492
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4124..4143)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4334..4355
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4518..4622
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4623..5483
                     /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(4841..4860)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5654..6242
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6143..6162
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6396..6413
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6530..6551
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6566..6596
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6604..6620
                     /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     6609..6631
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6628..6644
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6628..6644
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6663..6683
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6665..6683
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6711..6937
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6938..7118
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7165..7290
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7783..8016
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
ORIGIN
        1 ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata
       61 ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg
      121 aatagaagaa gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc
      181 tcgacggtat cgatcacgag actagcctcg agcggccgcc cccttcaccg agggcctatt
      241 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat
      301 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
      361 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
      421 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg
      481 tgtcttcgag gcttactaaa agccagataa cagtatgcgt atttgcgcgc tgatttttgc
      541 ggtataagaa tatatactga tatgtatacc cgaagtatgt caaaaagagg tgtgctatga
      601 agcagcgtat tacagtgaca gttgacagcg acagctatca gttgctcaag gcatatatga
      661 tgtcaatatc tccggtctgg taagcacaac catgcagaat gaagcccgtc gtctgcgtgc
      721 cgaacgctgg aaagcggaaa atcaggaagg gatggctgag gtcgcccggt ttattgaaat
      781 gaacggctct tttgctgacg agaacaggga ctggtgaaat gcagtttaag gtttacacct
      841 ataaaagaga gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc
      901 ccgggcgacg gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc
      961 gtgaacttta cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata
     1021 tggccagtgt gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa
     1081 atgacatcaa aaacgccatt aacctgatgt tctggggaat ataaaccctg caggacgaag
     1141 acccgtttga gagctaccgg agcagacgat atggcgtcgc tccggtagca agttcaaata
     1201 aggctagtcc gttatcaact tggagcagac gatatggcgt cgctccaagt ggcaccgagt
     1261 cggtgctttt tttgaattct cgacctcgag acaaatggca gtattcatcc acaattttaa
     1321 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
     1381 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta
     1441 ttacagggac agcagagatc cactttggcc gcggctcgag ggggttgggg ttgcgccttt
     1501 tccaaggcag ccctgggttt gcgcagggac gcggctgctc tgggcgtggt tccgggaaac
     1561 gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc gtcacccgga
     1621 tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc tgctccgccc ctaagtcggg
     1681 aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca cgtctcacta
     1741 gtaccctcgc agacggacag cgccagggag caatggcagc gcgccgaccg cgatgggctg
     1801 tggccaatag cggctgctca gcagggcgcg ccgagagcag cggccgggaa ggggcggtgc
     1861 gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg tgttccgcat
     1921 tctgcaagcc tccggagcgc acgtcggcag tcggctccct cgttgaccga atcaccgacc
     1981 tctctcccca gggggatcca ccggatcaag cttgccagct ggggcgccct ctggtaaggt
     2041 tgggaagccc tgcagatctg atcccggggg gcaatgagat atgaaaaagc ctgaactcac
     2101 cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca
     2161 gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt
     2221 cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt
     2281 tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattta gcgagagcct
     2341 gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagatctgc ctgaaaccga
     2401 actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct
     2461 tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg
     2521 gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga
     2581 cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga
     2641 ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga
     2701 caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata
     2761 cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg
     2821 ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct
     2881 ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc
     2941 ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac
     3001 acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga
     3061 tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagagta gatgccgacc
     3121 gggatctatc gataccgtcg acctcgaggg ggggcccggt acctttaaga ccaatgactt
     3181 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
     3241 ttcactccca acgaagaaaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc
     3301 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     3361 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     3421 gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt tcatgtcatc
     3481 ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt
     3541 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     3601 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     3661 tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     3721 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     3781 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta
     3841 cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt
     3901 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     3961 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     4021 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4081 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     4141 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4201 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     4261 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4321 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     4381 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     4441 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     4501 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     4561 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     4621 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     4681 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     4741 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     4801 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     4861 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     4921 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     4981 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     5041 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     5101 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     5161 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     5221 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     5281 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     5341 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     5401 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     5461 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     5521 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     5581 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5641 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5701 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5761 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     5821 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     5881 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     5941 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6001 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6061 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6121 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6181 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6241 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     6301 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     6361 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     6421 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     6481 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     6541 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     6601 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
     6661 gcgcaattaa ccctcactaa agggaacaaa agctggagct gcaagcttaa tgtagtctta
     6721 tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg ccttacaagg
     6781 agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc gtgccttatt
     6841 aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc cgcattgcag
     6901 agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt tagaccagat
     6961 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     7021 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     7081 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg
     7141 aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc
     7201 acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc
     7261 tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga
     7321 tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta
     7381 tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa
     7441 ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt
     7501 agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa
     7561 gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca
     7621 cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     7681 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     7741 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     7801 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     7861 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     7921 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     7981 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     8041 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     8101 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     8161 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     8221 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     8281 tataaaatta ttcataatga tagtaggagg ctt
//