LOCUS       sequence-319291-        6792 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for EGxxFP assay with PIGA intron 5 and exon 6 sequences.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6792)
  AUTHORS   Hyodo T, Rahman ML, Karnan S, Ito T, Toyoda A, Ota A, Wahiduzzaman 
            M, Tsuzuki S, Okada Y, Hosokawa Y, Konishi H
  TITLE     Tandem Paired Nicking Promotes Precise Genome Editing with Scarce 
            Interference by p53.
  JOURNAL   Cell Rep. 2020 Jan 28;30(4):1195-1207.e7. doi: 
            10.1016/j.celrep.2019.12.064.
  PUBMED    31995758
REFERENCE   2  (bases 1 to 6792)
  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"; doi: 
            "10.1016/j.celrep.2019.12"; journalName: "Cell Rep"; date: 
            "2020-01-28- 28"; volume: "30"; issue: "4"; pages: "1195-1207.e7"
COMMENT     Sequence Label: pCAG-EGxxFP-PIGA
FEATURES             Location/Qualifiers
     source          1..6792
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        17..396
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        398..673
                     /label=chicken beta-actin promoter
     intron          674..1688
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1614..1636
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1696..1715
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1743..1752
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1749..1751,1752..1754,1755..2267)
                     /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(1794..1815)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3322..3343
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3472..3491)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3537..3592
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3591..3610)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3947..3966)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3948..3966)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3976..3993)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3976..3992)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3985..4007)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4134..4589
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4221..4240)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4431..4452
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4615..4719
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4720..4788,4789..5580)
                     /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(4938..4957)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5751..6339
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6240..6259
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6493..6510
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6627..6648
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6663..6668,6669..6686,6687..6693)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6701..6717
                     /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)."
     primer_bind     6706..6728
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6725..6741
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6725..6741
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6760..6780
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6762..6780
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 gagctcatcg atactcgaca ttgattattg actagttatt aatagtaatc aattacgggg
       61 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      121 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      181 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      241 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      301 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
      361 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
      421 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
      481 ttattttgtg cagcgatggg ggcggggggg gggggggsgc gcgccaggcg gggcggggcg
      541 gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc
      601 gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag
      661 cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc
      721 ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga
      781 cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct
      841 gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc
      901 ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg
      961 cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg
     1021 gagcgcggcc gggggcggtg ccccgcggtg cggggggggc tgcgagggga acaaaggctg
     1081 cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca
     1141 accccccctg cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct
     1201 ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc aggtgggggt
     1261 gccgggcggg gcggggccgc ctcgggccgg ggagggctcg ggggaggggc gcggcggccc
     1321 ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc
     1381 gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag
     1441 gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga
     1501 aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc
     1561 tcggggctgt ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg
     1621 cttctggcgt gtgaccggcg gctctagcct ctgctaacca tgttcatgcc ttcttctttt
     1681 tcctacagct cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaatcta
     1741 gagccgccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     1801 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     1861 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     1921 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     1981 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     2041 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     2101 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     2161 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     2221 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     2281 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     2341 acaaccactg aggatccgct agcctgcagg tcgacgaatt cgatatcatt actatgacaa
     2401 caacaacaaa acacaagcaa cttttgtttg tatttgttta taaaatgttc ctgcaggatc
     2461 gggtcattgt ttatcatggg acaggtgatg gggtgatggg tgaaagtgct cacacttggc
     2521 ttctctccct ccaggtatat gaccgggtat cagtggaagc tgtgttgcca atggacaaac
     2581 gactggacag acttatttct cactgcggcc cagtaacagg ctacatcttt gctttgttgg
     2641 cagttttcaa cttcctcttc ctcattttct tgagatggat gactccagat tctatcattg
     2701 atgttgcaat agatgccact gggccacggg gtgcctggac taataactat tctcacagta
     2761 aaagaggggg tgagaataat gagatatcgg caagctgacc ctgaagttca tctgcaccac
     2821 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     2881 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     2941 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     3001 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     3061 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     3121 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     3181 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     3241 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     3301 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     3361 tctcggcatg gacgagctgt acaagtaact cgagactcct caggtgcagg ctgcctatca
     3421 gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag atctttttcc
     3481 ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata
     3541 aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag
     3601 gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc
     3661 aacatatgcc catatgctgg ctgccatgaa caaaggttgg ctataaagag gtcatcagta
     3721 tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga cttgaggtta
     3781 gatttttttt atattttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt
     3841 acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc
     3901 tcttctctta tgaagatccc tcgacttaat taaggtaccc aattcgccct atagtgagtc
     3961 gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     4021 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     4081 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
     4141 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     4201 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     4261 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     4321 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     4381 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     4441 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     4501 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     4561 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     4621 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     4681 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     4741 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     4801 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     4861 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     4921 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     4981 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5041 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     5101 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     5161 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     5221 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     5281 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     5341 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     5401 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     5461 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     5521 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     5581 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     5641 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     5701 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     5761 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     5821 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     5881 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     5941 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6001 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6061 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     6121 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     6181 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     6241 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     6301 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     6361 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     6421 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     6481 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     6541 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     6601 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     6661 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     6721 cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg
     6781 gaacaaaagc tg
//
