LOCUS       sequence-213814-        7180 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of i53 and a BFP reporter in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7180)
  AUTHORS   Yumlu S, Bashir S, Stumm J, Kuhn R
  TITLE     Efficient Gene Editing of Human Induced Pluripotent Stem Cells Using
            CRISPR/Cas9.
  JOURNAL   Methods Mol Biol. 2019;1961:137-151. doi: 
            10.1007/978-1-4939-9170-9_10.
  PUBMED    30912045
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: "Methods Mol
            Biol."; date: "2019"; volume: "1961:137-151. doi"; pages: " 
            10.1007/978-1-4939-9170-9_10"
COMMENT     Sequence Label: pCAG-i53-bpA-EF1BFP
FEATURES             Location/Qualifiers
     source          1..7180
                     /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..1713
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1637..1659
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1726..1745
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1726..1744
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1994..2010)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2030..2254
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     intron          2475..3413
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3370..3390
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3448..3457
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3454..3456,3457..3459,3460..4173)
                     /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(3499..3520)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    4204..4315
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4433..4450)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4433..4449)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4442..4464)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(4590..5045)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4727..4748)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4939..4958
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5072..5176
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5177..5245,5246..6037)
                     /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(5395..5414)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6208..6796
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6697..6716
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6950..6967
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7084..7105
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7120..7125,7126..7143,7144..7150)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7158..7174
                     /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(7163..7180,1..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 tcgctgcgcg ctgccttcgc
      721 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
      781 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gcgcttggtt
      841 taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct ccgggagggc
      901 cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt ggggagcgcc
      961 gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt
     1021 gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg gtgcgggggg
     1081 ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg agcagggggt
     1141 gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc ctccccgagt tgctgagcac
     1201 ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg cggggctcgc cgtgccgggc
     1261 ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc cggggagggc
     1321 tcgggggagg ggcgcggcgg cccccggagc gccggcggct gtcgaggcgc ggcgagccgc
     1381 agccattgcc ttttatggta atcgtgcgag agggcgcagg gacttccttt gtcccaaatc
     1441 tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg cggggcgaag
     1501 cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc gccgcgccgc
     1561 cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg ccttcggggg
     1621 ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag agcctctgct
     1681 aaccatgttc atgccttctt ctttttccta cagatcctta attaataata cgactcacta
     1741 taggggccac catgttgatt ttcgtgaaaa cccttaccgg gaaaaccatc accctcgagg
     1801 ttgaaccctc ggatacgata gaaaatgtaa aggccaagat ccaggataag gaaggaattc
     1861 ctcctgatca gcagagactg gcctttgctg gcaaatcgct ggaagatgga cgtactttgt
     1921 ctgactacaa tattctaaag gactctaaac ttcatcctct gttgagactt cgttgaacgc
     1981 gtaaatgatt gcagatccac tagttctaga gctcgctgat cagcctcgac tgtgccttct
     2041 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     2101 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     2161 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     2221 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     2281 ggctcgagat ccactagttc tagcctcgag gctagagcgg ccccggtgcc tagagaaggt
     2341 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
     2401 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
     2461 ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt
     2521 atggcccttg cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg
     2581 agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc
     2641 ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc
     2701 accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac
     2761 ctgctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca
     2821 ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat
     2881 gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag
     2941 ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg
     3001 caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg
     3061 ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca
     3121 cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc
     3181 gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt
     3241 ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta
     3301 ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt
     3361 ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt
     3421 gagcgatcgc atcgacagtg tttaaacgcc accatggtga gcaagggcga ggagctgttc
     3481 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     3541 gtgaggggcg agggcgaggg cgatgccacc aacggcaagc tgaccctgaa gttcatctgc
     3601 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgag ccacggcgtg
     3661 cagtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     3721 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcac ctacaagacc
     3781 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcgtc
     3841 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaactt caacagccac
     3901 aacatctata tcatggccgt caagcagaag aacggcatca aggtgaactt caagatccgc
     3961 cacaacgtgg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     4021 ggcgacggcc ccgtgctgct gcccgacagc cactacctga gcacccagtc cgtgctgagc
     4081 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttccgcac cgccgccggg
     4141 atcactctcg gcatggacga gctgtacaag tgagaattcc tagagctcgc tgatcagcct
     4201 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     4261 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     4321 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     4381 attgggaaga gaatagcagg catgcgttta aacattagtc gacagggccg ccactggccg
     4441 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     4501 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     4561 aacagttgcg cagcctgaat ggcgaatgga aattgtaagc gttaatattt tgttaaaatt
     4621 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     4681 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     4741 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     4801 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     4861 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     4921 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     4981 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     5041 cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     5101 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     5161 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     5221 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     5281 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     5341 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     5401 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     5461 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     5521 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     5581 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     5641 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     5701 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     5761 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     5821 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     5881 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     5941 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     6001 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     6061 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     6121 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     6181 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     6241 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     6301 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     6361 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     6421 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     6481 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     6541 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     6601 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     6661 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     6721 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     6781 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     6841 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     6901 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     6961 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     7021 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     7081 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     7141 tcgtatgttg tgtggaattg tgagcggata acaatttcac
//
