LOCUS       Exported                8309 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Photoactivatable transcription system. Lentiviral expression of 
            NANOG sgRNA4. Also contains a CMV-puro-t2A-mCherry expression 
            cassette.
ACCESSION   .
VERSION     .
KEYWORDS    sgRNA4_NANOG
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8309)
  AUTHORS   Nihongaki Y, Yamamoto S, Kawano F, Suzuki H, Sato M
  TITLE     CRISPR-Cas9-based Photoactivatable Transcription System.
  JOURNAL   Chem Biol. 2015 Feb 19;22(2):169-74. doi: 
            10.1016/j.chembiol.2014.12.011. Epub 2015 Jan 22.
  PUBMED    25619936
REFERENCE   2  (bases 1 to 8309)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8309
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    209..242
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     246..266
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        350..425
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        550..853
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        854..1057
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             1091..1687
                     /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(1091..1110)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1587..1607
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1697..1750
                     /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      1757..1766
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1763..2473
                     /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(1908..1926)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2176..2195
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    2493..2526
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    2582..3170
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2635..2655)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3053..3064)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3173..3189)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3174..3190)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             3699..3879
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(3941..4529)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4021..4040)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4700..5560)
                     /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     5323..5342
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5561..5665)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5749..5768)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5940..6319
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6321..6519
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             6537..6717
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6764..6889
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7388..7621
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7806..7850
                     /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    8148..8265
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 gggcccgctc tagagatccg acgcgccatc tctaggcccg cgccggcccc ctcgcacaga
       61 cttgtgggag aagctcggct actcccctgc cccggttaat ttgcatataa tatttcctag
      121 taactataga ggcttaatgt gcgataaaag acagataatc tgttcttttt aatactagct
      181 acattttaca tgataggctt ggatttctat aacttcgtat agcatacatt atacgaagtt
      241 ataaacagca caaaaggaaa ctcaccctaa ctgtaaagta attgtgtgtt ttgagactat
      301 aagtatccct tggagaacca ccttgttggc atattcctga tttaaaagtg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
      421 ggtgcttttt ttctcgagta ctaggatcca ttaggcggcc gcgtggataa ccgtattacc
      481 gccatgcatt agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
      541 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
      601 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
      661 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      721 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
      781 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      841 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
      901 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
      961 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
     1021 taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatccg
     1081 ctagcccacc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc
     1141 ccgggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt
     1201 cgacccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt
     1261 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac
     1321 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga
     1381 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg
     1441 gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa
     1501 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc
     1561 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac
     1621 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc
     1681 cggtgccgga tcgggagagg gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa
     1741 tcctggccca ccggtcgcca ccatggtgag caagggcgag gaggataaca tggccatcat
     1801 caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga
     1861 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
     1921 gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg
     1981 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc
     2041 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac
     2101 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa
     2161 cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga
     2221 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa
     2281 ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca
     2341 gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta
     2401 caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga
     2461 gctgtacaag taagaattcg tcgagggacc taataacttc gtatagcata cattatacga
     2521 agttatacat gtttaagggt tccggttcca ctaggtacaa ttcgatatca agcttatcga
     2581 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     2641 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     2701 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     2761 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     2821 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     2881 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     2941 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     3001 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     3061 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     3121 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg
     3181 tcgacctcga tcgagaccta gaaaaacatg gagcaatcac aagtagcaat acagcagcta
     3241 ccaatgctga ttgtgcctgg ctagaagcac aagaggagga ggaggtgggt tttccagtca
     3301 cacctcaggt acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt
     3361 taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa gatatccttg
     3421 atctgtggat ctaccacaca caaggctact tccctgattg gcagaactac acaccagggc
     3481 cagggatcag atatccactg acctttggat ggtgctacaa gctagtacca gttgagcaag
     3541 agaaggtaga agaagccaat gaaggagaga acacccgctt gttacaccct gtgagcctgc
     3601 atgggatgga tgacccggag agagaagtat tagagtggag gtttgacagc cgcctagcat
     3661 ttcatcacat ggcccgagag ctgcatccgg actgtactgg gtctctctgg ttagaccaga
     3721 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
     3781 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     3841 ccctcagacc cttttagtca gtgtggaaaa tctctagcag catgtgagca aaaggccagc
     3901 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3961 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4021 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4081 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4141 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4201 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4261 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4321 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4381 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4441 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4501 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4561 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4621 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4681 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4741 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4801 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4861 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4921 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     4981 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5041 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5101 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5161 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5221 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5281 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5341 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5401 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5461 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5521 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5581 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5641 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgcttaag
     5701 cggtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
     5761 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
     5821 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
     5881 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
     5941 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
     6001 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     6061 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     6121 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     6181 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     6241 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     6301 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     6361 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     6421 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     6481 gggcggtagg cgtgtacggt gggaggtcta tataagcagc gcgttttgcc tgtactgggt
     6541 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     6601 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     6661 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg
     6721 gcgcccgaac agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac
     6781 tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa
     6841 aattttgact agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg
     6901 ggggagaatt agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata
     6961 taaattaaaa catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg
     7021 cctgttagaa acatcagaag gctgtagaca aatactggga cagctacaac catcccttca
     7081 gacaggatca gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca
     7141 tcaaaggata gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa
     7201 caaaagtaag accaccgcac agcaagcggc cggccgcgct gatcttcaga cctggaggag
     7261 gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac
     7321 cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag
     7381 tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag
     7441 cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga
     7501 acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca
     7561 tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc
     7621 tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta
     7681 gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca
     7741 gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc
     7801 aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt
     7861 ttaacataac aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg
     7921 taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt
     7981 caccattatc gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa
     8041 tagaagaaga aggtggagag agagacagag acagatccat tcgattagtg aacggatcgg
     8101 cactgcgtgc gccaattctg cagacaaatg gcagtattca tccacaattt taaaagaaaa
     8161 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata
     8221 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg
     8281 gacagcagag atccagtttg gttagtacc
//
