LOCUS       Exported                8115 bp ds-DNA     circular UNA 29-JUL-2025
DEFINITION  Ligation of 2 sequences.
ACCESSION   urn.local...bm-jgygosj
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 8115)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Jul 30, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8115
                     /organism="unspecified"
                     /mol_type="genomic DNA"
     source          1129..5030
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          5108..5138
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    1..43
                     /standard_name="Annealed oligos for no-U6 (XbaI, PciI)
                     (concatenated sequence 1)"
                     /label=Geneious type: Concatenated sequence
                     /note="Geneious type: Concatenated sequence"
     misc_feature    1..4
                     /standard_name="Ligation"
                     /label=Ligation
                     /note="Geneious type: ligation"
     misc_feature    join(40..8115,1..4)
                     /standard_name="pKMW219__dSpRY_U6-EMX1-site1 (digested by
                     PciI, XbaI) Fragment 1 (concatenated sequence 2)"
                     /label=Geneious type: Concatenated sequence
                     /note="Geneious type: Concatenated sequence"
     misc_feature    join(40..8115,1..4)
                     /standard_name="pKMW090__SpRY_U6-reci extraction
                     (concatenated sequence 3)"
                     /label=Geneious type: Concatenated sequence
                     /note="Geneious type: Concatenated sequence"
     misc_feature    40..5111
                     /standard_name="pKMW084__SpRY-2A-Puro_U6-reci (digested by
                     EcoRI, FseI) Fragment 3 extraction (concatenated sequence 
                     1)"
                     /label=Geneious type: Concatenated sequence
                     /note="Geneious type: Concatenated sequence"
     misc_feature    40..43
                     /standard_name="Ligation"
                     /label=Ligation
                     /note="Geneious type: ligation"
     regulatory      338..615
                     /standard_name="chicken beta-actin promoter"
                     /label=chicken beta-actin promoter
                     /note="Geneious type: promoter"
     misc_feature    821..1158
                     /standard_name="Amp 130/131 (concatenated sequence 2)"
                     /label=Amp 130/131 (concatenated sequence 2)
                     /note="Geneious type: Concatenated sequence"
     misc_feature    821..850
                     /standard_name="Ligation"
                     /label=Ligation
                     /note="Geneious type: ligation"
     CDS             865..930
                     /codon_start=1
                     /standard_name="3xFLAG"
                     /product="three tandem FLAG epitope tags, followed by an 
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             937..957
                     /codon_start=1
                     /standard_name="SV40 NLS"
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             982..5082
                     /standard_name="dSpRY"
                     /product="N-terminal portion of Streptococcus pyogenes
                     Cas9 (Zetsche et al., 2015)"
                     /label=dSpRY
     misc_feature    1007
                     /standard_name="A"
                     /label=A
                     /note="Geneious type: Editing History Replacement"
     misc_feature    1129..5030
                     /standard_name="Amp 132/133_SpRY (concatenated sequence
                     3)"
                     /label=Amp 132/133_SpRY (concatenated sequence
                     3)
                     /note="Geneious type: Concatenated sequence"
     misc_feature    1129..1158
                     /standard_name="Ligation"
                     /label=Ligation
                     /note="Geneious type: ligation"
     misc_feature    3496..3498
                     /standard_name="CAT"
                     /label=CAT
                     /note="Geneious type: Editing History Replacement"
     misc_feature    join(5001..5111,5135..8115,1..4,40..850)
                     /standard_name="pDAC177 (pSpCas9(BB)-2A-Puro V2.0)
                     (digested by XcmI, AgeI) Fragment 3 (concatenated sequence 
                     1)"
                     /label=Geneious type: Concatenated sequence
                     /note="Geneious type: Concatenated sequence"
     misc_feature    5001..5030
                     /standard_name="Ligation"
                     /label=Ligation
                     /note="Geneious type: ligation"
     CDS             5083..5130
                     /standard_name="nucleoplasmin NLS"
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
     misc_feature    5107^5108
                     /standard_name="ACGAAAAAGG"
                     /label=ACGAAAAAGG
                     /note="Geneious type: Editing History Deletion"
     misc_feature    5108..5138
                     /standard_name="KMW155/KMW156 Annealed (concatenated
                     sequence 2)"
                     /label=KMW155/KMW156 Annealed (concatenated
                     sequence 2)
                     /note="Geneious type: Concatenated sequence"
     misc_feature    5108..5111
                     /standard_name="Ligation"
                     /label=Ligation
                     /note="Geneious type: ligation"
     misc_feature    join(5135..8115,1..4)
                     /standard_name="pKMW084__SpRY-2A-Puro_U6-reci (digested by
                     EcoRI, FseI) Fragment 3 - pDAC177 (pSpCas9(BB)-2A-Puro 
                     V2.0) (digested by XcmI, AgeI) Fragment 3 (concatenated 
                     sequence 1) (concatenated sequence 3)"
                     /label=Geneious type: Concatenated sequence
                     /note="Geneious type: Concatenated sequence"
     misc_feature    5135..5138
                     /standard_name="Ligation"
                     /label=Ligation
                     /note="Geneious type: ligation"
     misc_feature    5138^5139
                     /standard_name="CCTA"
                     /label=CCTA
                     /note="Geneious type: Editing History Deletion"
     misc_feature    5167..5374
                     /standard_name="bGH poly(A) signal"
                     /label=bGH poly(A) signal
                     /note="Geneious type: polyA_signal"
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5598..6053
     regulatory      6335..6439
                     /gene="bla"
                     /standard_name="AmpR promoter"
                     /label=AmpR promoter
                     /note="Geneious type: promoter"
     CDS             6440..7300
                     /codon_start=1
                     /standard_name="AmpR"
                     /label=AmpR
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7471..8059
                     /direction=RIGHT
ORIGIN
        1 catgtttagc gcgtgcgcca attctgcaga caaatggctc tagaggtacc cgttacataa
       61 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
      121 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      181 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      241 ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt tcctacttgg
      301 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
      361 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
      421 ttattttgtg cagcgatggg ggcggggggg gggggggggc gcgcgccagg cggggcgggg
      481 cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc
      541 gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga
      601 agcgcgcggc gggcgggagt cgctgcgacg ctgccttcgc cccgtgcccc gctccgccgc
      661 cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg
      721 ggacggccct tctcctccgg gctgtaatta gctgagcaag aggtaagggt ttaagggatg
      781 gttggttggt ggggtattaa tgtttaatta cctggagcac ctgcctgaaa tcactttttt
      841 tcaggttgga ccggtgccac catggactat aaggaccacg acggagacta caaggatcat
      901 gatattgatt acaaagacga tgacgataag atggccccaa agaagaagcg gaaggtcggt
      961 atccacggag tcccagcagc cgacaagaag tacagcatcg gcctggccat cggcaccaac
     1021 tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg
     1081 ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac
     1141 agcggcgaaa cagccgagag aacccggctg aagagaaccg ccagaagaag atacaccaga
     1201 cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac
     1261 gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag
     1321 cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc
     1381 atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc
     1441 tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg
     1501 aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag
     1561 ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc
     1621 agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag
     1681 aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc
     1741 aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac
     1801 ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag
     1861 aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag
     1921 gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg
     1981 ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag
     2041 agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag
     2101 ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac
     2161 agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc
     2221 cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag
     2281 gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct
     2341 ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc
     2401 ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg
     2461 atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg
     2521 tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg
     2581 agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag
     2641 accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc
     2701 ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac
     2761 cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac
     2821 attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa
     2881 cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg
     2941 agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag
     3001 tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg
     3061 cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc
     3121 ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag
     3181 aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac
     3241 aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag
     3301 aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag
     3361 atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac
     3421 tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc
     3481 gactacgatg tggacgccat cgtgcctcag agctttctga aggacgactc catcgacaac
     3541 aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag
     3601 gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag
     3661 agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc
     3721 ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc
     3781 ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa
     3841 gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa
     3901 gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga
     3961 accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag
     4021 gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc
     4081 aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac
     4141 ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg
     4201 gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc
     4261 gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcag acccaagagg
     4321 aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc
     4381 ctgtggccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc
     4441 aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc
     4501 gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg
     4561 atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg
     4621 gcctctgcca agcagctgca gaagggaaac gaactggccc tgccctccaa atatgtgaac
     4681 ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag
     4741 aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc
     4801 gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac
     4861 aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc
     4921 ctgaccagac tgggagcccc tagagccttc aagtactttg acaccaccat cgaccccaag
     4981 cagtacagaa gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc
     5041 ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg
     5101 aaaaaggccg gccaggcaaa aaagaaaaag taagaattct aactagagct cgctgatcag
     5161 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     5221 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     5281 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     5341 aggattggga agagaatagc aggcatgctg gggagcggcc gcaggaaccc ctagtgatgg
     5401 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     5461 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     5521 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     5581 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     5641 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     5701 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     5761 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     5821 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     5881 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg
     5941 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     6001 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     6061 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     6121 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     6181 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     6241 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     6301 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     6361 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     6421 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     6481 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     6541 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     6601 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     6661 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     6721 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     6781 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     6841 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     6901 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     6961 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     7021 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     7081 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     7141 agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     7201 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     7261 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     7321 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     7381 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     7441 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     7501 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     7561 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     7621 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     7681 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     7741 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     7801 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     7861 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     7921 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     7981 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     8041 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     8101 ctggcctttt gctca
//
