LOCUS       Exported                6059 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Base plasmid for sgRNA cloning with Hygromycin resistance and Tol2 
            transposon arms.
ACCESSION   .
VERSION     .
KEYWORDS    sgBbsI (p2Tol-U6-2xBbsI-sgRNA-HygR)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6059)
  AUTHORS   Arbab M, Srinivasan S, Hashimoto T, Geijsen N, Sherwood RI
  TITLE     Cloning-free CRISPR.
  JOURNAL   Stem Cell Reports. 2015 Oct 27. pii: S2213-6711(15)00284-2. doi: 
            10.1016/j.stemcr.2015.09.022.
  PUBMED    26527385
REFERENCE   2  (bases 1 to 6059)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6059
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(7..462)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(144..165)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     356..375
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     589..611
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     603..620
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     604..620
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     630..649
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        630..648
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      878..887
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    893..926
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    964..997
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     promoter        1015..1255
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     1015..1035
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1186..1205
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     polyA_signal    1484..1605
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    1729..1850
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(1980..3005)
                     /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"
     promoter        complement(3054..3383)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3068..3203
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3122..3141)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(3875..3895)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3875..3893)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3914..3930)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3914..3930)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3927..3949)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3938..3954
                     /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(3962..3992)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4007..4028
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4145..4162)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4316..4904)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4396..4415)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5075..5935)
                     /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     5698..5717
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5936..6040)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
       61 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
      121 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
      181 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
      241 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
      301 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
      361 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
      421 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc
      481 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
      541 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
      601 gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta
      661 ccggcatatg gttcttgaca gaggtgtaaa aagtactcaa aaattttact caagtgaaag
      721 tacaagtact tagggaaaat tttactcaat taaaagtaaa agtatctggc tagaatctta
      781 cttgagtaaa agtaaaaaag tactccatta aaattgtact tgagtattaa ggaagtaaaa
      841 gtaaaagcaa gaaagatcga tctcgaagga tctggaggcc accatggtgt cgataacttc
      901 gtatagcata cattatacga agttatcgat agtccagtgt ggtggaattg cccttggccg
      961 gccataactt cgtataggat actttatacg aagttatggt accggcgcgc atgtgagggc
     1021 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
     1081 ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
     1141 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
     1201 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac
     1261 accgggtctt cgagaagacc tgtttaagag ctatgctgga aacagcatag caagtttaaa
     1321 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttgttttag
     1381 agctagaaat agcaagttaa aataaggcta gtccgttttt agcgcgtgcg ccaattctgc
     1441 agacaaatgg ctctagaggt acggccgctt cgagcagaca tgataagata cattgatgag
     1501 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     1561 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     1621 attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag caagtaaaac
     1681 ctctacaaat gtggtaaaat cgcgaaggtc gacggtatac agacatgata agatacattg
     1741 atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt
     1801 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt ggggtgggcg
     1861 aagaactcca gcatgagatc cccgcgctgg aggatcatcc agccggcgtc ccggaaaacg
     1921 attccgaagc ccaacctttc atagaaggcg gcggtggaat cgaaatctcg tagcacgtgc
     1981 tattcctttg ccctcggacg agtgctgggg cgtcggtttc cactatcggc gagtacttct
     2041 acacagccat cggtccagac ggccgcgctt ctgcgggcga tttgtgtacg cccgacagtc
     2101 ccggctccgg atcggacgat tgcgtcgcat cgaccctgcg cccaagctgc atcatcgaaa
     2161 ttgccgtcaa ccaagctctg atagagttgg tcaagaccaa tgcggagcat atacgcccgg
     2221 agccgcggcg atcctgcaag ctccggatgc ctccgctcga agtagcgcgt ctgctgctcc
     2281 atacaagcca accacggcct ccagaagaag atgttggcga cctcgtattg ggaatccccg
     2341 aacatcgcct cgctccagtc aatgaccgct gttatgcggc cattgtccgt caggacattg
     2401 ttggagccga aatccgcgtg cacgaggtgc cggacttcgg ggcagtcctc ggcccaaagc
     2461 atcagctcat cgagagcctg cgcgacggac gcactgacgg tgtcgtccat cacagtttgc
     2521 cagtgataca catggggatc agcaatcgcg catatgaaat cacgccatgt agtgtattga
     2581 ccgattcctt gcggtccgaa tgggccgaac ccgctcgtct ggctaagatc ggccgcagcg
     2641 atcgcatcca tggcctccgc gaccggctgc agaacagcgg gcagttcggt ttcaggcagg
     2701 tcttgcaacg tgacaccctg tgcacggcgg gagatgcaat aggtcaggct ctcgctgaat
     2761 tccccaatgt caagcacttc cggaatcggg agcgcggccg atgcaaagtg ccgataaaca
     2821 taacgatctt tgtagaaacc atcggcgcag ctatttaccc gcaggacata tccacgccct
     2881 cctacatcga agctgaaagc acgagattct tcgccctccg agagctgcat caggtcggag
     2941 acgctgtcga acttttcgat cagaaacttc tcgacagacg tcgcggtgag ttcaggcttt
     3001 ttcatcacgt gctgatcaga tccgaaaatg gatatacaag ctcccgggag ctttttgcaa
     3061 aagcctaggc ctccaaaaaa gcctcctcac tacttctgga atagctcaga ggcagaggcg
     3121 gcctcggcct ctgcataaat aaaaaaaatt agtcagccat ggggcggaga atgggcggaa
     3181 ctgggcggag ttaggggcgg gatgggcgga gttaggggcg ggactatggt tgctgactaa
     3241 ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt tccacacctg
     3301 gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac
     3361 tttccacacc ctaactgaca cacattccac agaattaatt cgcgttaaat ttttgttaaa
     3421 tcagctcatt ttttaaccaa taggccgaaa tcgatccggc tttagcatgc atgctttgca
     3481 tacttctgcc tgctggggag cctggggact ttccacacct ggttgctgac taattgagat
     3541 gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac cctaactgac
     3601 acacattcca cagaattcaa gtgatctcca aaaaataagt actttttgac tgtaaataaa
     3661 attgtaagga gtaaaaagta cttttttttc taaaaaaatg taattaagta aaagtaaaag
     3721 tattgatttt taattgtact caagtaaagt aaaaatcccc aaaaataata cttaagtaca
     3781 gtaatcaagt aaaattactc aagtacttta cacctctggt tcttgacccc ctaccttcag
     3841 caagcccagc agatccgagc tccagctttt gttcccttta gtgagggtta attgcgcgct
     3901 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     3961 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4021 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4081 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4141 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4201 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4261 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4321 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4381 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4441 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4501 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4561 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4621 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4681 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4741 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4801 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4861 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4921 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4981 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5041 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5101 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5161 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5221 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5281 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5341 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5401 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5461 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5521 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     5581 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     5641 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     5701 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     5761 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     5821 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     5881 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     5941 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6001 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtg
//
