LOCUS       sequence-205859-        4182 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  donor vector backbone with constitutive eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4182)
  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 4182)
  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..4182
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     56..75
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    142..166
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(172..174,175..177,178..888)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(172..174,175..177,178..888)
                     /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(217..238)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(478..497)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     825..846
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(950..974)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(989..1005)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(990..1006)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(1021..1038)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1027..1251
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(1307..1325)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1346..1362)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1346..1362)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1359..1381)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1370..1386
                     /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(1394..1400,1401..1418,1419..1424))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1439..1460
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1577..1594)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1748..2336)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1828..1847)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2507..3298,3299..3367))
                     /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     3130..3149
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3368..3472)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3498..3953)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3635..3656)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3847..3866
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4080..4102
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4094..4111
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4095..4111
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4118..4137
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4118..4136
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac
       61 gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tggatcctag tggatccccc
      121 gggctgcagg aattcgatat cacaagtttg tacaaaaaag caggctggac catggtgagc
      181 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
      241 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
      301 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
      361 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
      421 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
      481 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
      541 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
      601 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
      661 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
      721 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
      781 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
      841 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggg cggtggaaga
      901 tctgggaatt caaggcctct cgagcctcta gattctgcag ccctatagca cccagctttc
      961 ttgtacaaag tggtgatatc aagcttatcg ataccgtcga cctcgagggc cgctgatcag
     1021 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     1081 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     1141 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     1201 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     1261 cggaaagaac cagctggggc ttaattaaga gctccagctt ttgttccctt tagtgagggt
     1321 taatttcgag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     1381 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     1441 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     1501 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     1561 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     1621 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     1681 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     1741 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     1801 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     1861 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     1921 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     1981 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     2041 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     2101 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     2161 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     2221 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     2281 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     2341 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     2401 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     2461 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     2521 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     2581 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     2641 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     2701 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     2761 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     2821 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     2881 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     2941 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     3001 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     3061 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     3121 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     3181 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     3241 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     3301 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     3361 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     3421 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     3481 cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     3541 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     3601 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     3661 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     3721 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     3781 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     3841 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     3901 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc
     3961 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
     4021 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
     4081 ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga
     4141 attgggtacc ggccatgatg gccgaattaa ttcaagctag tc
//
