LOCUS       sequence-369376-        5765 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Base plasmid for sgRNA cloning with Hygromycin resistance and Tol2 
            transposon arms.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5765)
  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 5765)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Stem Cell 
            Reports. 2015 Oct 27. pii: S2213-6711(15)00284-2. doi: 
            10.1016/j.stemcr.2015.09.022."
COMMENT     Sequence Label: sgBbsI (p2Tol-U6-2xBbsI-sgRNA-HygR)
FEATURES             Location/Qualifiers
     source          1..5765
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(34..461)
                     /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     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(678..689,690..852)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     regulatory      877..886
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(892..925)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(963..996)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     promoter        1014..1262
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1014..1034
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1185..1204
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     polyA_signal    1483..1604
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    1728..1849
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(1979..3004)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(1979..3004)
                     /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(3053..3249)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(3061..3271)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3067..3202
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3121..3140)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     3295..3315
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     misc_feature    join(3325..3512,3513..3524)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3581..3601)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3581..3599)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3620..3636)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3620..3636)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3633..3655)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3644..3660
                     /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(join(3668..3674,3675..3692,3693..3698))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3713..3734
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3851..3868)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4022..4610)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4102..4121)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4781..5572,5573..5641))
                     /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     5404..5423
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5642..5746)
                     /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 ggcgggtgtg gtggtcacgc tgcgcgtaac
      421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac
      661 cggcatatgg ttcttgacag aggtgtaaaa agtactcaaa aattttactc aagtgaaagt
      721 acaagtactt agggaaaatt ttactcaatt aaaagtaaaa gtatctggct agaatcttac
      781 ttgagtaaaa gtaaaaaagt actccattaa aattgtactt gagtattaag gaagtaaaag
      841 taaaagcaag aaagatcgat ctcgaaggat ctggaggcca ccatggtgtc gataacttcg
      901 tatagcatac attatacgaa gttatcgata gtccagtgtg gtggaattgc ccttggccgg
      961 ccataacttc gtataggata ctttatacga agttatggta ccggcgcgca tgtgagggcc
     1021 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg
     1081 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
     1141 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
     1201 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca
     1261 ccgggtcttc gagaagacct gtttaagagc tatgctggaa acagcatagc aagtttaaat
     1321 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga
     1381 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca
     1441 gacaaatggc tctagaggta cggccgcttc gagcagacat gataagatac attgatgagt
     1501 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     1561 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     1621 ttcattttat gtttcaggtt cagggggaga tgtgggaggt tttttaaagc aagtaaaacc
     1681 tctacaaatg tggtaaaatc gcgaaggtcg acggtataca gacatgataa gatacattga
     1741 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     1801 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagttg gggtgggcga
     1861 agaactccag catgagatcc ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga
     1921 ttccgaagcc caacctttca tagaaggcgg cggtggaatc gaaatctcgt agcacgtgct
     1981 attcctttgc cctcggacga gtgctggggc gtcggtttcc actatcggcg agtacttcta
     2041 cacagccatc ggtccagacg gccgcgcttc tgcgggcgat ttgtgtacgc ccgacagtcc
     2101 cggctccgga tcggacgatt gcgtcgcatc gaccctgcgc ccaagctgca tcatcgaaat
     2161 tgccgtcaac caagctctga tagagttggt caagaccaat gcggagcata tacgcccgga
     2221 gccgcggcga tcctgcaagc tccggatgcc tccgctcgaa gtagcgcgtc tgctgctcca
     2281 tacaagccaa ccacggcctc cagaagaaga tgttggcgac ctcgtattgg gaatccccga
     2341 acatcgcctc gctccagtca atgaccgctg ttatgcggcc attgtccgtc aggacattgt
     2401 tggagccgaa atccgcgtgc acgaggtgcc ggacttcggg gcagtcctcg gcccaaagca
     2461 tcagctcatc gagagcctgc gcgacggacg cactgacggt gtcgtccatc acagtttgcc
     2521 agtgatacac atggggatca gcaatcgcgc atatgaaatc acgccatgta gtgtattgac
     2581 cgattccttg cggtccgaat gggccgaacc cgctcgtctg gctaagatcg gccgcagcga
     2641 tcgcatccat ggcctccgcg accggctgca gaacagcggg cagttcggtt tcaggcaggt
     2701 cttgcaacgt gacaccctgt gcacggcggg agatgcaata ggtcaggctc tcgctgaatt
     2761 ccccaatgtc aagcacttcc ggaatcggga gcgcggccga tgcaaagtgc cgataaacat
     2821 aacgatcttt gtagaaacca tcggcgcagc tatttacccg caggacatat ccacgccctc
     2881 ctacatcgaa gctgaaagca cgagattctt cgccctccga gagctgcatc aggtcggaga
     2941 cgctgtcgaa cttttcgatc agaaacttct cgacagacgt cgcggtgagt tcaggctttt
     3001 tcatcacgtg ctgatcagat ccgaaaatgg atatacaagc tcccgggagc tttttgcaaa
     3061 agcctaggcc tccaaaaaag cctcctcact acttctggaa tagctcagag gcagaggcgg
     3121 cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa tgggcggaac
     3181 tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt gctgactaat
     3241 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta
     3301 actgacacac attccacaga attcaagtga tctccaaaaa ataagtactt tttgactgta
     3361 aataaaattg taaggagtaa aaagtacttt tttttctaaa aaaatgtaat taagtaaaag
     3421 taaaagtatt gatttttaat tgtactcaag taaagtaaaa atccccaaaa ataatactta
     3481 agtacagtaa tcaagtaaaa ttactcaagt actttacacc tctggttctt gaccccctac
     3541 cttcagcaag cccagcagat ccgagctcca gcttttgttc cctttagtga gggttaattg
     3601 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     3661 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     3721 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3781 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3841 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3901 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3961 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4021 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4081 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4141 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4201 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4261 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4321 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4381 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4441 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4501 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4561 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4621 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4681 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4741 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4801 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4861 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4921 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4981 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5041 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5101 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5161 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5221 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5281 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5341 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5401 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5461 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5521 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5581 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5641 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5701 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5761 aagtg
//
