LOCUS       sequence-205860-        5801 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  donor vector backbone with constitutive eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5801)
  AUTHORS   Kim SI, Matsumoto T, Kagawa H, Nakamura M, Hirohata R, Ueno A, 
            Ohishi M, Sakuma T, Soga T, Yamamoto T, Woltjen K
  TITLE     Microhomology-assisted scarless genome editing in human iPSCs.
  JOURNAL   Nat Commun. 2018 Mar 5;9(1):939. doi: 10.1038/s41467-018-03044-y.
  PUBMED    29507284
REFERENCE   2  (bases 1 to 5801)
  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: "Nat 
            Commun."; date: "2018-03-5"; volume: "9(1):939. doi"; pages: " 
            10.1038/s41467-018-03044-y"
COMMENT     Sequence Label: pCAG-eGFP-pA
FEATURES             Location/Qualifiers
     source          1..5801
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(20..448)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(130..151)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     342..361
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     575..597
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     589..606
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     590..606
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     613..632
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        613..631
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        678..1057
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1060..1335
                     /label=chicken beta-actin promoter
     intron          1336..2344
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2268..2290
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2352..2371
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    2438..2462
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(2468..2470,2471..2473,2474..3184)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2468..2470,2471..2473,2474..3184)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2513..2534)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2774..2793)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3121..3142
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3246..3270)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3285..3301)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3286..3302)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(3317..3334)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3323..3547
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(3603..3621)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3642..3658)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3642..3658)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3655..3677)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3666..3682
                     /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)."
     promoter        complement(join(3690..3696,3697..3714,3715..3720))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3735..3756
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3873..3890)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4044..4632)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4124..4143)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4803..5594,5595..5663))
                     /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     5426..5445
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5664..5768)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
       61 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
      121 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
      181 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
      241 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
      301 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
      361 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
      421 gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg
      481 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
      541 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
      601 gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtaccggcca tgatggccga
      661 attaattcaa gctagtcgac attgattatt gactagttat taatagtaat caattacggg
      721 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      781 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      841 agtaacgcca atagggactt tccattgacg tcaatgggtg gactatttac ggtaaactgc
      901 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
      961 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
     1021 gcagtacatc tacgtattag tcatcgctat taccatgggt cgaggtgagc cccacgttct
     1081 gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt
     1141 aattattttg tgcagcgatg ggggcggggg gggggggggc gcgcgccagg cggggcgggg
     1201 cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc
     1261 gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga
     1321 agcgcgcggc gggcgggagt cgctgcgttg ccttcgcccc gtgccccgct ccgcgccgcc
     1381 tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac
     1441 ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggctcgt ttcttttctg
     1501 tggctgcgtg aaagccttaa agggctccgg gagggccctt tgtgcggggg ggagcggctc
     1561 ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggcccgc gctgcccggc
     1621 ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcgtgtg cgcgagggga
     1681 gcgcggccgg gggcggtgcc ccgcggtgcg ggggggctgc gaggggaaca aaggctgcgt
     1741 gcggggtgtg tgcgtggggg ggtgagcagg gggtgtgggc gcggcggtcg ggctgtaacc
     1801 cccccctgca cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc
     1861 gtgcggggcg tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc
     1921 cgggcggggc ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccg
     1981 gagcgccggc ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg
     2041 cgagagggcg cagggacttc ctttgtccca aatctggcgg agccgaaatc tgggaggcgc
     2101 cgccgcaccc cctctagcgg gcgcgggcga agcggtgcgg cgccggcagg aaggaaatgg
     2161 gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccatctc cagcctcggg
     2221 gctgccgcag ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg
     2281 gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct
     2341 acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattgga
     2401 tcctagtgga tcccccgggc tgcaggaatt cgatatcaca agtttgtaca aaaaagcagg
     2461 ctggaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     2521 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     2581 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     2641 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     2701 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     2761 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     2821 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     2881 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     2941 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     3001 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     3061 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     3121 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     3181 caagggcggt ggaagatctg ggaattcaag gcctctcgag cctctagatt ctgcagccct
     3241 atagcaccca gctttcttgt acaaagtggt gatatcaagc ttatcgatac cgtcgacctc
     3301 gagggccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     3361 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     3421 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     3481 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     3541 tctatggctt ctgaggcgga aagaaccagc tggggcttaa ttaagagctc cagcttttgt
     3601 tccctttagt gagggttaat ttcgagcttg gcgtaatcat ggtcatagct gtttcctgtg
     3661 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     3721 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     3781 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     3841 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     3901 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     3961 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4021 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4081 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4141 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4201 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4261 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4321 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4381 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4441 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     4501 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4561 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4621 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4681 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4741 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     4801 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     4861 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     4921 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     4981 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5041 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5101 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5161 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5221 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5281 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5341 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     5401 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     5461 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     5521 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     5581 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     5641 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     5701 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     5761 gttccgcgca catttccccg aaaagtgcca cctaaattgt a
//
