LOCUS       sequence-236898-        6360 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning plasmid for EGxFP assay with self-targeting gRNA-s.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6360)
  AUTHORS   Talas A, Huszar K, Kulcsar PI, Varga JK, Varga E, Toth E, Welker Z, 
            Erdos G, Pach PF, Welker A, Gyorgypal Z, Tusnady GE, Welker E
  TITLE     A method for characterizing Cas9 variants via a one-million target 
            sequence library of self-targeting sgRNAs.
  JOURNAL   Nucleic Acids Res. 2021 Jan 15. pii: 6101605. doi: 
            10.1093/nar/gkaa1220.
  PUBMED    33450024
REFERENCE   2  (bases 1 to 6360)
  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: "Nucleic 
            Acids Res. 2021 Jan 15. pii: 6101605. doi: 10.1093/nar/gkaa1220."
COMMENT     Sequence Label: pAT-9222
FEATURES             Location/Qualifiers
     source          1..6360
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        389..664
                     /label=chicken beta-actin promoter
     intron          665..1671
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1597..1619
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1679..1698
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1726..1735
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1732..1734,1735..1737,1738..2250)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIE"
     primer_bind     complement(1777..1798)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     promoter        2336..2584
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2336..2356
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2507..2526
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2700..2718
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     rep_origin      complement(2803..3391)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2883..2902)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4003..4024
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4153..4172)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4218..4273
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4272..4291)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4628..4647)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4629..4647)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4657..4674)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4657..4673)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4666..4688)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4815..5270
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4902..4921)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5112..5133
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5296..5400
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5401..5469,5470..6261)
                     /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     complement(5619..5638)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gatactcgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
       61 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      121 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      181 atagggactt tccattgacg tcaatgggtg gactatttac ggtaaactgc ccacttggca
      241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      361 tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc
      421 cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt
      481 gcagcgatgg gggcgggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg
      541 gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
      601 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
      661 ggcgggagtc gctgcgttgc cttcgccccg tgccccgctc cgcgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggctcgtt tcttttctgt ggctgcgtga
      841 aagccttaaa gggctccggg agggcccttt gtgcgggggg gagcggctcg gggggtgcgt
      901 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggcccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcgtgtgc gcgaggggag cgcggccggg
     1021 ggcggtgccc cgcggtgcgg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt
     1081 gcgtgggggg gtgagcaggg ggtgtgggcg cggcggtcgg gctgtaaccc ccccctgcac
     1141 ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg tgcggggcgt
     1201 ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg
     1261 gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggccccgg agcgccggcg
     1321 gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc
     1381 agggacttcc tttgtcccaa atctggcgga gccgaaatct gggaggcgcc gccgcacccc
     1441 ctctagcggg cgcgggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc
     1501 cttcgtgcgt cgccgcgccg ccgtcccctt ctccatctcc agcctcgggg ctgccgcagg
     1561 gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg
     1621 gcggctctag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc
     1681 aacgtgctgg ttattgtgct gtctcatcat tttggcaaat ctagagccgc catggtgagc
     1741 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     1801 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     1861 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1921 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     1981 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     2041 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     2101 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     2161 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     2221 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     2281 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctgaggaggg
     2341 cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta gagagataat
     2401 tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta
     2461 ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct
     2521 taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa
     2581 caccgggtct tcggatccag atctgaagac ctgggttaga gctagaaata gcaagttaac
     2641 ctaaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttgcatgc
     2701 tgtctcttat acacatctcc gagcccacga gaccgatgat ctcatgtgag caaaaggcca
     2761 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     2821 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     2881 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     2941 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     3001 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3061 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3121 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3181 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3241 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     3301 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     3361 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     3421 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgatcg gcaagctgac
     3481 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     3541 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     3601 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     3661 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     3721 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     3781 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     3841 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     3901 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     3961 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     4021 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaac tcgagactcc
     4081 tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc tggctcacaa
     4141 ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga
     4201 gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt
     4261 tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga
     4321 gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga acaaaggttg
     4381 gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag
     4441 aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt
     4501 aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact
     4561 actcccagtc atagctgtcc ctcttctctt atgaagatcc ctcgacttaa ttaaggtacc
     4621 caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg
     4681 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     4741 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     4801 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4861 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     4921 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     4981 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     5041 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     5101 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     5161 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     5221 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     5281 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5341 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5401 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5461 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5521 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5581 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5641 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5701 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5761 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5821 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5881 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5941 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     6001 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     6061 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     6121 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6181 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6241 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6301 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
//
