LOCUS       sequence-134230-        6062 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses palindromic sgRNA for use in self-cloning CRISPR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6062)
  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 6062)
  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: sgPal1 (p2Tol-U6-sgPal1-HygR)
FEATURES             Location/Qualifiers
     source          1..6062
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(34..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"
     misc_feature    join(679..690,691..853)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     regulatory      878..887
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(893..926)
                     /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(964..997)
                     /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        1015..1263
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     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    1487..1608
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    1732..1853
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(1983..3008)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(1983..3008)
                     /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(3057..3386)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3071..3206
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3125..3144)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     misc_feature    join(3622..3809,3810..3821)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3878..3898)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3878..3896)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3917..3933)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3917..3933)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3930..3952)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3941..3957
                     /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(3965..3971,3972..3989,3990..3995))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4010..4031
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4148..4165)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4319..4907)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4399..4418)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5078..5869,5870..5938))
                     /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     5701..5720
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5939..6043)
                     /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 accgctctgt gactagtcac agaggtttaa gagctatgct ggaaacagca tagcaagttt
     1321 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttgttt
     1381 tagagctaga aatagcaagt taaaataagg ctagtccgtt tttagcgcgt gcgccaattc
     1441 tgcagacaaa tggctctaga ggtacggccg cttcgagcag acatgataag atacattgat
     1501 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     1561 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     1621 tgcattcatt ttatgtttca ggttcagggg gagatgtggg aggtttttta aagcaagtaa
     1681 aacctctaca aatgtggtaa aatcgcgaag gtcgacggta tacagacatg ataagataca
     1741 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     1801 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttggggtgg
     1861 gcgaagaact ccagcatgag atccccgcgc tggaggatca tccagccggc gtcccggaaa
     1921 acgattccga agcccaacct ttcatagaag gcggcggtgg aatcgaaatc tcgtagcacg
     1981 tgctattcct ttgccctcgg acgagtgctg gggcgtcggt ttccactatc ggcgagtact
     2041 tctacacagc catcggtcca gacggccgcg cttctgcggg cgatttgtgt acgcccgaca
     2101 gtcccggctc cggatcggac gattgcgtcg catcgaccct gcgcccaagc tgcatcatcg
     2161 aaattgccgt caaccaagct ctgatagagt tggtcaagac caatgcggag catatacgcc
     2221 cggagccgcg gcgatcctgc aagctccgga tgcctccgct cgaagtagcg cgtctgctgc
     2281 tccatacaag ccaaccacgg cctccagaag aagatgttgg cgacctcgta ttgggaatcc
     2341 ccgaacatcg cctcgctcca gtcaatgacc gctgttatgc ggccattgtc cgtcaggaca
     2401 ttgttggagc cgaaatccgc gtgcacgagg tgccggactt cggggcagtc ctcggcccaa
     2461 agcatcagct catcgagagc ctgcgcgacg gacgcactga cggtgtcgtc catcacagtt
     2521 tgccagtgat acacatgggg atcagcaatc gcgcatatga aatcacgcca tgtagtgtat
     2581 tgaccgattc cttgcggtcc gaatgggccg aacccgctcg tctggctaag atcggccgca
     2641 gcgatcgcat ccatggcctc cgcgaccggc tgcagaacag cgggcagttc ggtttcaggc
     2701 aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc aataggtcag gctctcgctg
     2761 aattccccaa tgtcaagcac ttccggaatc gggagcgcgg ccgatgcaaa gtgccgataa
     2821 acataacgat ctttgtagaa accatcggcg cagctattta cccgcaggac atatccacgc
     2881 cctcctacat cgaagctgaa agcacgagat tcttcgccct ccgagagctg catcaggtcg
     2941 gagacgctgt cgaacttttc gatcagaaac ttctcgacag acgtcgcggt gagttcaggc
     3001 tttttcatca cgtgctgatc agatccgaaa atggatatac aagctcccgg gagctttttg
     3061 caaaagccta ggcctccaaa aaagcctcct cactacttct ggaatagctc agaggcagag
     3121 gcggcctcgg cctctgcata aataaaaaaa attagtcagc catggggcgg agaatgggcg
     3181 gaactgggcg gagttagggg cgggatgggc ggagttaggg gcgggactat ggttgctgac
     3241 taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac
     3301 ctggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg
     3361 gactttccac accctaactg acacacattc cacagaatta attcgcgtta aatttttgtt
     3421 aaatcagctc attttttaac caataggccg aaatcgatcc ggctttagca tgcatgcttt
     3481 gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct gactaattga
     3541 gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca caccctaact
     3601 gacacacatt ccacagaatt caagtgatct ccaaaaaata agtacttttt gactgtaaat
     3661 aaaattgtaa ggagtaaaaa gtactttttt ttctaaaaaa atgtaattaa gtaaaagtaa
     3721 aagtattgat ttttaattgt actcaagtaa agtaaaaatc cccaaaaata atacttaagt
     3781 acagtaatca agtaaaatta ctcaagtact ttacacctct ggttcttgac cccctacctt
     3841 cagcaagccc agcagatccg agctccagct tttgttccct ttagtgaggg ttaattgcgc
     3901 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     3961 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     4021 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4081 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     4141 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4201 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4261 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4321 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4381 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4441 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4501 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4561 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4621 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4681 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4741 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4801 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4861 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4921 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4981 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5041 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5101 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5161 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5221 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5281 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5341 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     5401 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5461 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5521 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5581 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5641 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     5701 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5761 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5821 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     5881 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     5941 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6001 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6061 tg
//
