LOCUS       sequence-224969-        7175 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 7175)
  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 7175)
  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..7175
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..18
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2..18
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        38..417
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        419..696
                     /label=chicken beta-actin promoter
     intron          697..1708
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1632..1654
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1721..1740
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1721..1739
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1989..2005)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2025..2249
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     intron          2470..3408
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3365..3385
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3443..3452
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3449..3451,3452..3454,3455..4168)
                     /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(3494..3515)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    4199..4310
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4428..4445)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4428..4444)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4437..4459)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4586..5041
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4673..4692)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4883..4904
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5067..5171
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5172..5240,5241..6032)
                     /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(5390..5409)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6203..6791
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6692..6711
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6945..6962
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7079..7100
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7115..7120,7121..7138,7139..7145)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7153..7169
                     /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     join(7158..7175,1..5)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 acaggaaaca gctatgacca tgaggcgcgc cggattcgac attgattatt gactagttat
       61 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
      121 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      181 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      241 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      301 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
      361 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc
      421 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
      481 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg
      541 cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg
      601 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      661 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgtc gcgccttcgc
      721 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
      781 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gcgcttggtt
      841 taatgacggc tcgtttcttt tctgtggctg cgtgaaagcc ttaaagggct ccgggagggc
      901 cctttgtgcg ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc
      961 cgcgtgcggc ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt
     1021 gcgctccgcg tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg
     1081 ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt
     1141 gggcgcggcg gtcgggctgt aacccccccc tgcacccccc tccccgagtt gctgagcacg
     1201 gcccggcttc gggtgcgggg ctccgtgcgg ggcgtggcgc ggggctcgcc gtgccgggcg
     1261 gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc ggggagggct
     1321 cgggggaggg gcgcggcggc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag
     1381 ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg
     1441 gcggagccga aatctgggag gcgccgccgc accccctcta gcgggcgcgg gcgaagcggt
     1501 gcggcgccgg caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc
     1561 cccttctcca tctccagcct cggggctgcc gcagggggac ggctgccttc gggggggacg
     1621 gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct ctgctaacca
     1681 tgttcatgcc ttcttctttt tcctacagat ccttaattaa taatacgact cactataggg
     1741 gccaccatgt tgattttcgt gaaaaccctt accgggaaaa ccatcaccct cgaggttgaa
     1801 ccctcggata cgatagaaaa tgtaaaggcc aagatccagg ataaggaagg aattcctcct
     1861 gatcagcaga gactggcctt tgctggcaaa tcgctggaag atggacgtac tttgtctgac
     1921 tacaatattc taaaggactc taaacttcat cctctgttga gacttcgttg aacgcgtaaa
     1981 tgattgcaga tccactagtt ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     2041 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     2101 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     2161 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     2221 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     2281 gagatccact agttctagcc tcgaggctag agcggccccg gtgcctagag aaggtggcgc
     2341 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
     2401 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
     2461 agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac gggttatggc
     2521 ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg tgattcttga tcccgagctt
     2581 cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt
     2641 gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt
     2701 cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg atgacctgct
     2761 gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct gcacactggt
     2821 atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg
     2881 gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc tcaagctggc
     2941 cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg
     3001 ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca
     3061 gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc acccacacaa
     3121 aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga gtaccgggcg
     3181 ccgtccaggc acctcgatta gttctcgagc ttttggagta cgtcgtcttt aggttggggg
     3241 gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga agttaggcca
     3301 gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg atcttggttc
     3361 attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt gtcgtgagcg
     3421 atcgcatcga cagtgtttaa acgccaccat ggtgagcaag ggcgaggagc tgttcaccgg
     3481 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgag
     3541 gggcgagggc gagggcgatg ccaccaacgg caagctgacc ctgaagttca tctgcaccac
     3601 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgagccacg gcgtgcagtg
     3661 cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     3721 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcacctaca agacccgcgc
     3781 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcgtcgactt
     3841 caaggaggac ggcaacatcc tggggcacaa gctggagtac aacttcaaca gccacaacat
     3901 ctatatcatg gccgtcaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     3961 cgtggaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     4021 cggccccgtg ctgctgcccg acagccacta cctgagcacc cagtccgtgc tgagcaaaga
     4081 ccccaacgag aagcgcgatc acatggtcct gctggagttc cgcaccgccg ccgggatcac
     4141 tctcggcatg gacgagctgt acaagtgaga attcctagag ctcgctgatc agcctcgact
     4201 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     4261 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     4321 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     4381 gaagagaata gcaggcatgc gtttaaacat tagtcgacag ggccgccact ggccgtcgtt
     4441 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     4501 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     4561 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4621 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     4681 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     4741 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     4801 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     4861 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     4921 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     4981 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     5041 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     5101 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5161 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     5221 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     5281 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     5341 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     5401 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     5461 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     5521 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     5581 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     5641 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     5701 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     5761 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     5821 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     5881 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     5941 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     6001 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     6061 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6121 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6181 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6241 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6301 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     6361 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6421 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6481 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6541 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     6601 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     6661 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     6721 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     6781 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     6841 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     6901 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     6961 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     7021 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     7081 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     7141 tgttgtgtgg aattgtgagc ggataacaat ttcac
//
