LOCUS       Exported                5190 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Empty backbone for cloning sgRNA sequence to be used in nanoblades 
            system (Optimized for increased genome editing efficiency via Chen B
            et al., 2013).
ACCESSION   .
VERSION     .
KEYWORDS    SUPERBLADE5
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5190)
  AUTHORS   Mangeot PE, Risson V, Fusil F, Marnef A, Laurent E, Blin J, 
            Mournetas V, Massourides E, Sohier TJM, Corbin A, Aube F, Teixeira 
            M, Pinset C, Schaeffer L, Legube G, Cosset FL, Verhoeyen E, Ohlmann 
            T, Ricci EP
  TITLE     Genome editing in primary cells and in vivo using viral-derived 
            Nanoblades loaded with Cas9-sgRNA ribonucleoproteins.
  JOURNAL   Nat Commun. 2019 Jan 3;10(1):45. doi: 10.1038/s41467-018-07845-z.
  PUBMED    30604748
REFERENCE   2  (bases 1 to 5190)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5190
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(3..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     674..690
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     674..690
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        705..945
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     705..725
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     876..895
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        complement(3002..3020)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3041..3057)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3041..3057)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3054..3076)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3065..3081
                     /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(3089..3119)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3134..3155
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3272..3289)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3443..4031)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3523..3542)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4202..5062)
                     /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     4825..4844
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5063..5167)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg agctccaccg
      661 cggtggcggc cgctctagaa ctagtggatc cgaggggacc cagagagggc ctatttccca
      721 tgattccttc atatttgcat atacgataca aggctgttag agagataatt agaattaatt
      781 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
      841 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt
      901 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac accggagacg
      961 gttgtaaatg agcacacaaa atacacatgc taaaatatta tattctatga cctttataaa
     1021 atcaaccaaa atcttctttt taataacttt agtatcaata attagaattt ttatgttcct
     1081 ttttgcaaac ttttaataaa aatgagcaaa ataaaaaaac gctagtttta gtaactcgcg
     1141 ttgttttctt cacctttaat aatagctact ccaccacttg ttcctaagcg gtcagctcct
     1201 gcttcaatca ttttttgagc atcttcaaat gttctaactc caccagctgc tttaactaaa
     1261 gcattgtctt taacaactga cttcattagt ttaacatctt caaatgttgc acctgatttt
     1321 gaaaatcctg ttgatgtttt aacaaattct aatccagctt caacagctat ttcacaagct
     1381 ttcatgattt cttcttttgt taataaacaa ttttccataa tacatttaac aacatgtgat
     1441 ccagctgctt tttttacagc tttcatgtct tctaaaacta attcataatt tttgtctttt
     1501 aatgcaccaa tatttaatac catatcaatt tctgttgcac catctttaat tgcttcagaa
     1561 acttcgaatg cttttgtagc tgttgtgcat gcacctagag gaaaacctac aacatttgtt
     1621 attcctacat ttgtgccttt taataattct ttacaatagc ttgttcaata tgaattaaca
     1681 caaactgttg caaaatcaaa ttcaattgct tcatcacata attgtttaat ttcagctttc
     1741 gtagcatctt gttttaataa tgtgtgatct atatatttgt ttagtttcat tttttctcct
     1801 atatattcat ttttaatttt aattctttaa taatttcgtc tactttaact ttagcgtatt
     1861 gaacagattc accaacacct ataaaataaa tttttagttt aggttcagtt ccacttgggc
     1921 gaacagcaaa tcatgactta tcttctaaat aaaattttag taagtcttgt cctggcatat
     1981 tatacattcc atcgatgtag tcttcaacat taacaacttt aagtccagca atttgagtta
     2041 agggtgttgc tctcaatgat ttcattaatg gttcaatttt taatttcttt tcttctggtt
     2101 taaaattcaa gtttaaagtg aaagtgtaat atgcacccat ttctttaaat aaatcttcta
     2161 aatagtctac taatgtttta ttttgttttt tataaaatca agcagcctct gctattaata
     2221 tagaagcttg tattccatct ttatctctag ctgagtcatc aattacatat ccataacttt
     2281 cttcataagc aaaaacaaaa tttaatccgt tatcttcttc tttagcaatt tctctaccca
     2341 ttcatttaaa tccagttaaa gtttttacaa tattaactcc atatttttca tgagcgattc
     2401 tatcacccaa atcacttgtt acaaaacttg aatatagagc cggatttttt ggaatgctat
     2461 ttaagcgttt tagatttgat aattttcaat caattaaaat tggtcctgtt tgatttccat
     2521 ctaatcttac aaaatgacca tcatgtttta ttgccattcc aaatctgtca gcatctgggt
     2581 cattcataat aataatatct gcatcatgtt taataccata ttcaagcggt atttttcatg
     2641 caggatcaaa ttctggattt ggatttacaa catttttaaa tgtttcatct tcaaatgcat
     2701 gctcttcaac ctcaataacg ttatatcctg attcacgtaa tatttttggg gtaaatttag
     2761 ttcctgttcc attaactgcg ctaaaaataa tttttaaatc ttttttagct tcttgctctt
     2821 ttttgtacgt ctctgtttaa gagctatgct ggaaacagca tagcaagttt aaataaggct
     2881 agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttgaat tctagatctt
     2941 gagacaaatg gcagtattca tccacaattc tcgagggggg gcccggtacc agcttttgtt
     3001 ccctttagtg agggttaatt tcgagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     3061 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     3121 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     3181 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     3241 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     3301 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     3361 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     3421 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3481 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3541 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3601 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     3661 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     3721 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     3781 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     3841 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     3901 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     3961 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4021 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4081 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4141 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4201 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4261 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4321 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     4381 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     4441 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4501 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4561 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     4621 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     4681 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     4741 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     4801 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     4861 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     4921 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     4981 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5041 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5101 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5161 ttccgcgcac atttccccga aaagtgccac
//
