LOCUS       sequence-243693-        6368 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 6368)
  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 6368)
  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..6368
                     /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..1679
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1605..1627
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1687..1706
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1734..1743
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1740..1742,1743..1745,1746..2258)
                     /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(1785..1806)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     promoter        2344..2592
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2344..2364
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2515..2534
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2708..2726
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     rep_origin      complement(2811..3399)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2891..2910)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4011..4032
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4161..4180)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4226..4281
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4280..4299)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4636..4655)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4637..4655)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4665..4682)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4665..4681)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4674..4696)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4823..5278
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4910..4929)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5120..5141
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5304..5408
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5409..5477,5478..6269)
                     /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(5627..5646)
                     /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 gagtatttac 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 ggggggggsg cgcgccrggs ggggsggggs ggggsgrggg
      541 gsggggsggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
      601 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
      661 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
      721 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
      781 tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg
      841 tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggggtgc
      901 gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga
      961 gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc
     1021 cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg
     1081 tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct
     1141 gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg
     1201 gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg
     1261 ggcggggccg cctcgggccg gggagggctc gggggagggg cgcggcggcc cccggagcgc
     1321 cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag
     1381 ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg
     1441 caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg
     1501 ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg
     1561 tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg
     1621 tgtgaccggc ggctctagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc
     1681 tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaatct agagccgcca
     1741 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     1801 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     1861 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     1921 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     1981 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     2041 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     2101 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     2161 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     2221 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     2281 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     2341 gaggagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
     2401 gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
     2461 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
     2521 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
     2581 ggacgaaaca ccgggtcttc ggatccagat ctgaagacct gggttagagc tagaaatagc
     2641 aagttaacct aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     2701 ttgcatgctg tctcttatac acatctccga gcccacgaga ccgatgatct catgtgagca
     2761 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     2821 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     2881 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     2941 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     3001 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     3061 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     3121 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     3181 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     3241 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     3301 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     3361 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     3421 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgatcggc
     3481 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     3541 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     3601 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     3661 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     3721 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     3781 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     3841 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     3901 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     3961 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     4021 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaactc
     4081 gagactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg
     4141 gctcacaaat accactgaga tctttttccc tctgccaaaa attatgggga catcatgaag
     4201 ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt
     4261 tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat
     4321 cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc tgccatgaac
     4381 aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta
     4441 ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt
     4501 ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc
     4561 tcctgactac tcccagtcat agctgtccct cttctcttat gaagatccct cgacttaatt
     4621 aaggtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta
     4681 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     4741 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     4801 cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     4861 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     4921 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     4981 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     5041 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     5101 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     5161 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     5221 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     5281 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     5341 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     5401 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     5461 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     5521 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     5581 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     5641 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     5701 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     5761 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     5821 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     5881 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     5941 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     6001 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     6061 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     6121 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     6181 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     6241 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     6301 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     6361 atctcatc
//
