LOCUS       Exported                9060 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses gRNA HL1 gR4 in the pSB700 backbone.
ACCESSION   .
VERSION     .
KEYWORDS    pSB700 HL1gR4
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9060)
  AUTHORS   Smith CJ, Castanon O, Said K, Volf V, Khoshakhlagh P, Hornick A, 
            Ferreira R, Wu CT, Guell M, Garg S, Ng AHM, Myllykallio H, Church GM
  TITLE     Enabling large-scale genome editing at repetitive elements by 
            reducing DNA nicking.
  JOURNAL   Nucleic Acids Res. 2020 May 21;48(9):5183-5195. doi: 
            10.1093/nar/gkaa239.
  PUBMED    32315033
REFERENCE   2  (bases 1 to 9060)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9060
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /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    2028..2143
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     promoter        2246..2486
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     2246..2266
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2417..2436
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2516..2591
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        2665..2968
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          3244..4260
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4184..4206
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4268..4287
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      4288..4297
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             4294..5004
                     /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
                     EGHHSTGGMDELYK"
     primer_bind     complement(4439..4457)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4707..4726
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    5028..5616
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5081..5101)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5499..5510)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5823..6456
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6585..6601)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6585..6601)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6598..6620)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6609..6625
                     /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)."
     promoter        complement(6633..6663)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6678..6699
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6816..6833)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6987..7575)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7067..7086)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7746..8606)
                     /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     8369..8388
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8607..8711)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8759..8893
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8796..8815)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8850..8869
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag
     2041 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     2101 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     2161 acagcagaga tccagtttat cattagtgaa cggatctcga cggtatcgat cacgagacta
     2221 gcctcgagcg gccgccccct tcaccgaggg cctatttccc atgattcctt catatttgca
     2281 tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat
     2341 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     2401 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
     2461 ggctttatat atcttgtgga aaggacgaaa caccgattct accagaggta caagggtttt
     2521 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
     2581 cgagtcggtg ctttttttga attctcgacc tcgagacaaa tggcagtatt cgtcattagt
     2641 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     2701 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     2761 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     2821 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     2881 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     2941 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct
     3001 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
     3061 tgcagcgatg ggggcggggg gggggggcgc gcgccaggcg gggcggggcg gggggggggg
     3121 gggggggggg gggggggggg gggggcggcg gcagccaatc agagcggcgc gctccgaaag
     3181 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
     3241 gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc
     3301 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct
     3361 cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt
     3421 gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg
     3481 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag
     3541 cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc
     3601 gggggcggtg ccccgcggtg cggggggggc tgcgagggga acaaaggctg cgtgcggggt
     3661 gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca accccccctg
     3721 cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg
     3781 cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc aggtgggggt gccgggcggg
     3841 gcggggccgc ctcgggccgg ggggggctcg ggggaggggc gcggcggccc ccggagcgcc
     3901 ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg
     3961 gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag gcgccgccgc
     4021 accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg
     4081 gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt
     4141 ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt
     4201 gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag
     4261 ctcctgggca acgtgctggt tattgtggcc accatggtga gcaagggcga agaagataac
     4321 atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc
     4381 cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc
     4441 aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag
     4501 ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag
     4561 ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg
     4621 gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg
     4681 cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag
     4741 gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg
     4801 ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag
     4861 aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac
     4921 aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccacca ctccaccggc
     4981 ggcatggacg agctgtacaa gtaatgtaca agtaaagcgt ctggaacaat caacctctgg
     5041 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     5101 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     5161 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     5221 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     5281 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     5341 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     5401 attccgtggt gttgtcgggg aagctgacgt cctttccatg gctgctcgcc tgtgttgcca
     5461 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     5521 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     5581 agacgagtcg gatctccctt tgggccgcct ccccgcctgg aattaattct gcagtcgaga
     5641 cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc
     5701 ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt
     5761 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagagggg
     5821 actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt ggatctacca
     5881 cacacaaggc tacttccctg attagcagaa ctacacacca gggccagggg tcagatatcc
     5941 actgaccttt ggatggtgct acaagctagt accagttgag ccagataagg tagaagaggc
     6001 caataaagga gagaacacca gcttgttaca ccctgtgagc ctgcatggga tggatgaccc
     6061 ggagagagaa gtgttagagt ggaggtttga cagccgccta gcatttcatc acgtggcccg
     6121 agagctgcat ccggagtact tcaagaactg ctgatatcga gcttgctaca agggactttc
     6181 cgctggggac tttccaggga ggcgtggcct gggcgggact ggggagtggc gagccctcag
     6241 atcctgcata taagcagctg ctttttgcct gtactgggtc tctctggtta gaccagatct
     6301 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     6361 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     6421 tcagaccctt ttagtcagtg tggaaaatct ctagcagtag tagttcatgt catcttatta
     6481 ttcagtattt ataacttgca aagaaatgaa tatcagagag tgagaggcct tgacattgct
     6541 agcgttttac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     6601 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     6661 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     6721 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     6781 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     6841 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     6901 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     6961 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     7021 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     7081 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     7141 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     7201 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     7261 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     7321 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     7381 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     7441 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     7501 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     7561 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     7621 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     7681 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     7741 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     7801 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     7861 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     7921 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     7981 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     8041 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     8101 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     8161 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     8221 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     8281 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     8341 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     8401 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     8461 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     8521 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     8581 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     8641 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     8701 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatcaa
     8761 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     8821 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     8881 tcatgtctgg atcaactgga taactcaagc taaccaaaat catcccaaac ttcccacccc
     8941 ataccctatt accactgcca attacctgtg gtttcattta ctctaaacct gtgattcctc
     9001 tgaattattt tcattttaaa gaaattgtat ttgttaaata tgtactacaa acttagtagt
//
