LOCUS       sequence-233159-        7180 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of i53 (53BP1) inhibitor and BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7180)
  AUTHORS   Bashir S, Dang T, Rossius J, Wolf J, Kuhn R
  TITLE     Enhancement of CRISPR-Cas9 induced precise gene editing by targeting
            histone H2A-K15 ubiquitination.
  JOURNAL   BMC Biotechnol. 2020 Oct 23;20(1):57. doi: 
            10.1186/s12896-020-00650-x.
  PUBMED    33097066
REFERENCE   2  (bases 1 to 7180)
  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: "BMC 
            Biotechnol."; date: "2020-10-23"; volume: "20(1):57. doi"; pages: " 
            10.1186/s12896-020-00650-x"
COMMENT     Sequence Label: pCAG-i53
FEATURES             Location/Qualifiers
     source          1..7180
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          162..1178
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1102..1124
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1191..1210
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1191..1209
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1459..1475)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    1495..1719
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     intron          1940..2878
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2835..2855
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      2913..2922
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2919..2921,2922..2924,2925..3638)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(2964..2985)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    3669..3780
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3898..3915)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3898..3914)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3907..3929)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(4055..4510)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4192..4213)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4404..4423
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4537..4641
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4642..4710,4711..5502)
                     /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(4860..4879)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5673..6261
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6162..6181
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6415..6432
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6549..6570
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6585..6590,6591..6608,6609..6615)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6623..6639
                     /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     6628..6650
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6647..6663
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6647..6663
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        6683..7062
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(7064..7180,1..161)
                     /label=chicken beta-actin promoter
ORIGIN
        1 gggggcgcgc gccrggsggg gsggggsggg gsgrggggsg gggsggggcg aggcggagag
       61 gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc
      121 ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc
      181 ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg
      241 cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct
      301 tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag gggctccggg
      361 agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga
      421 gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc
      481 tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg
      541 gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag
      601 ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc cgagttgctg
      661 agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc
      721 cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct cgggccgggg
      781 rgggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga
      841 gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc
      901 aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg
      961 cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc
     1021 gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac ggctgccttc
     1081 gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct
     1141 ctgctaacca tgttcatgcc ttcttctttt tcctacagat ccttaattaa taatacgact
     1201 cactataggg gccaccatgt tgattttcgt gaaaaccctt accgggaaaa ccatcaccct
     1261 cgaggttgaa ccctcggata cgatagaaaa tgtaaaggcc aagatccagg ataaggaagg
     1321 aattcctcct gatcagcaga gactggcctt tgctggcaaa tcgctggaag atggacgtac
     1381 tttgtctgac tacaatattc taaaggactc taaacttcat cctctgttga gacttcgttg
     1441 aacgcgtaaa tgattgcaga tccactagtt ctagagctcg ctgatcagcc tcgactgtgc
     1501 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     1561 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     1621 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     1681 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     1741 gctggggctc gagatccact agttctagcc tcgaggctag agcggccccg gtgcctagag
     1801 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga
     1861 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg
     1921 gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac
     1981 gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg tgattcttga
     2041 tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc
     2101 ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg
     2161 gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg
     2221 atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct
     2281 gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg
     2341 cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc
     2401 tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg
     2461 ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg
     2521 ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc
     2581 acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga
     2641 gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta cgtcgtcttt
     2701 aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga
     2761 agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg
     2821 atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt
     2881 gtcgtgagcg atcgcatcga cagtgtttaa acgccaccat ggtgagcaag ggcgaggagc
     2941 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     3001 tcagcgtgag gggcgagggc gagggcgatg ccaccaacgg caagctgacc ctgaagttca
     3061 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgagccacg
     3121 gcgtgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     3181 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcacctaca
     3241 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     3301 gcgtcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aacttcaaca
     3361 gccacaacat ctatatcatg gccgtcaagc agaagaacgg catcaaggtg aacttcaaga
     3421 tccgccacaa cgtggaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     3481 ccatcggcga cggccccgtg ctgctgcccg acagccacta cctgagcacc cagtccgtgc
     3541 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc cgcaccgccg
     3601 ccgggatcac tctcggcatg gacgagctgt acaagtgaga attcctagag ctcgctgatc
     3661 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     3721 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     3781 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     3841 ggaggattgg gaagagaata gcaggcatgc gtttaaacat tagtcgacag ggccgccact
     3901 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     3961 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     4021 ttcccaacag ttgcgcagcc tgaatggcga atggaaattg taagcgttaa tattttgtta
     4081 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     4141 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     4201 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     4261 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     4321 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     4381 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     4441 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     4501 cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     4561 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     4621 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     4681 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     4741 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     4801 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     4861 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     4921 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     4981 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     5041 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     5101 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     5161 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     5221 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     5281 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     5341 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     5401 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     5461 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     5521 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     5581 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     5641 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     5701 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     5761 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     5821 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     5881 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     5941 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6001 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6061 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     6121 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     6181 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     6241 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     6301 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     6361 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     6421 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     6481 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     6541 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     6601 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     6661 gaccatgagg cgcgccggat tcgacattga ttattgacta gttattaata gtaatcaatt
     6721 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     6781 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     6841 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     6901 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     6961 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     7021 acttggcagt acatctacgt attagtcatc gctattacca tggtcgaggt gagccccacg
     7081 ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt
     7141 ttttaattat tttgtgcagc gatgggggcg gggggggggg
//
