LOCUS       sequence-217828-        7086 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid for knock-in T2A-H2B-eGFP to the c-terminal of mouse 
            Pdgfra coding sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7086)
  AUTHORS   Gu B, Posfai E, Rossant J
  TITLE     Efficient generation of targeted large insertions by microinjection 
            into two-cell-stage mouse embryos.
  JOURNAL   Nat Biotechnol. 2018 Jun 11. pii: nbt.4166. doi: 10.1038/nbt.4166.
  PUBMED    29889212
REFERENCE   2  (bases 1 to 7086)
  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 
            Biotechnol. 2018 Jun 11. pii: nbt.4166. doi: 10.1038/nbt.4166."
COMMENT     Sequence Label: Pdgfra-T2A-H2B-eGFP donor
FEATURES             Location/Qualifiers
     source          1..7086
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1581..1634
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1641..2018
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      2031..2040
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2037..2039,2040..2042,2043..2756)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2037..2039,2040..2042,2043..2753)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2082..2103)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2343..2362)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2690..2711
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2758..2774)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2758..2774)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3829..3848)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3830..3848)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3858..3875)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3858..3874)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3867..3889)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(4015..4470)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4152..4173)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4364..4383
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4497..4601
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4602..4670,4671..5462)
                     /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(4820..4839)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5633..6221
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6122..6141
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6375..6392
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6509..6530
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6545..6550,6551..6568,6569..6575)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6583..6599
                     /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     6588..6610
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6607..6623
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6607..6623
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6642..6662
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6644..6662
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     6691..6707
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     6692..6708
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ggtggggatt ttgggcaggc tggtgatttt agggatagta acactaacac ctaacacttt
       61 tgttttaaaa atgtttagta cacagtagca gtgagtacac ctcccaatta gtgggtacag
      121 gggttgacat ttgccccatt ctctttatcc agattggtgt cacataaatc atcatgacta
      181 cagacacaca cacacacaca cacacacaca cacacacaca ccttatttct cacccagtga
      241 ctgaaagcta agtagaaaac accaagataa ttcattccta gtattctgcg acactctttc
      301 ctaagaatac gatcattcat gctgtgtcta tcatgaccac acggaagaga gtgaactgtt
      361 aaagaaaatt agagttggag gtatagaatt catgttggcc atatctgagg ccataggttc
      421 acatccctgt actgaaagaa aaaagagaag aagaaaagga agccaacaac cggtctcaga
      481 ttcatttcat acctgggctt tattcaaatt cagttgaata ataactgcta atttattgag
      541 ctctttatat gtttatagtt atcagattat tatcgtattt aatcatctca gagatcttta
      601 tttttatttt tattatgtat attttacttt tgagattata tttatgtgtg tgtgtacaga
      661 tctgtacacc taagtatgtg actgcaaccg gcttagactg tatgatactc gtatgtgtac
      721 gttttcgggg ctgaccattt ggtattggat agccaatttg gtgtattcgt ccctggggaa
      781 ggcccttttt ctggttctca accttccttc gttatctgtc gctcttggtc tagaaaagag
      841 gctgcctgag ctccttgctt acccattacc gtgcctattg gtgccgttct ggtgcaggtc
      901 ttgcgggcaa ggcttctatg ccttgtgtaa atggtaaact gaagactttt ctcttctggg
      961 cctttagacc cagaggggcg gggtgaacaa gaggcagggg cctggcagga agtgaaggaa
     1021 gtgtgggatc ccatccttct gcttcctaag gttttttttt tcccttctct ctcagtacct
     1081 tttgattggc tgttgcttgt tctctcccct gataggctga ggagggaaag tagaaactct
     1141 tactcttgtt agccacgccc acaaaagacc cctggcagga aaggcagcaa actgacctgc
     1201 gcagttggcc ctgttttggt tttgtgttgg ctctgtgcag caatggccag tgagtggctg
     1261 gctggctcag ccgctcactg gccatgctcg gccactcatg cttcaacagg aaaggacctt
     1321 gtgcatgagg aaagagtccc gctgcttcag gcagccaccc tggaaatgca gccccgtagg
     1381 cagtctgtcc atgaccttag cctttgcttt tcaatcccat cagctcacag acttcggaag
     1441 agagtgccat cgagacaggt tccagtagtt ccaccttcat caagagagag gacgagacca
     1501 tcgaggacat cgacatgatg gatgacattg gtatagattc ctcggacctg gtggaggaca
     1561 gcttcctgga tatcgaattc gagggcagag gaagtcttct aacatgcggt gacgtggagg
     1621 agaatcccgg ccctaccggt atgccagagc cagcgaagtc tgctcccgcc ccgaaaaagg
     1681 gctccaagaa ggcggtgact aaggcgcaga agaaaggcgg caagaagcgc aagcgcagcc
     1741 gcaaggagag ctattccatc tatgtgtaca aggttctgaa gcaggtccac cctgacaccg
     1801 gcatttcgtc caaggccatg ggcatcatga attcgtttgt gaacgacatt ttcgagcgca
     1861 tcgcaggtga ggcttcccgc ctggcgcatt acaacaagcg ctcgaccatc acctccaggg
     1921 agatccagac ggccgtgcgc ctgctgctgc ctggggagtt ggccaagcac gccgtgtccg
     1981 agggtactaa ggccatcacc aagtacacca gcgctaagga tccaccggtc gccaccatgg
     2041 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     2101 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     2161 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     2221 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     2281 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     2341 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     2401 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     2461 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     2521 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     2581 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     2641 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     2701 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaaggat
     2761 ccactagttc tagagcggcc gctaactgac acgctccggg tatcatcttc tccgggagcc
     2821 tcctcgggat aaccttaaga aaaccacttc attgcaatgc aaaagttgag aagaggactt
     2881 gggtgatgtg gagagagaaa gttcccgagg ccgagggcct tggtaagcct gtgtggatga
     2941 ctgggatact tggattgaac tttatccgtg ttgcctcttg caacgcgtca gtagcatctc
     3001 agcggcgtgt gcggtttgga aatggatgga caagagagtg actggccaca gaagacgggc
     3061 tttgtgtttc cagggcatgg gtgagagtcc cacacgcgcc aacttgtact gcagacaact
     3121 ccagcattgt aattatgtaa ataactctta accgaggatg ggtttagatc tgtgttaact
     3181 ctcctctccg gacctctgaa gagaccactc agtccacccg tgtgcttcct tccttcttgg
     3241 aatccggtac cagctgctgt tgaattttct catgaagtac atgcaaaacc actttgaact
     3301 ttggaaggta ctggaaccgt cgtgttttac tgtcttgtaa gtgttaaaga ggcaaaaaga
     3361 taaaaactaa ccaaccgtga gtagtaggtt tgggtctttt agggcaccca agaattcctc
     3421 tgtatcttgt aatctgtact tataacacta ctactgttta acaagaatgc taaatatgta
     3481 atccgtaaca tgatttcttt acagagaaag cacaattaaa acaaaaccct tactaagaag
     3541 gtgacaagtt tggcagtttt tgacatttat attaagtaac acgtttctca ataaagtata
     3601 ataggtttag cggattaaag ttagcaaaca ctagagagca gagtgtaagt agttatccag
     3661 aaagtcaaac ctttttgttt tgttctaagc agtctcccta atccaaggca ctaggaacag
     3721 ttaatgacct ctgaagtact gggataagca aagcagaaac ccggtgggtg tcctaagtgt
     3781 tctcagtaga aaggcgtgcg gccgccaccg cggtggagct ccaattcgcc ctatagtgag
     3841 tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     3901 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     3961 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggaaattg
     4021 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     4081 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     4141 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     4201 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     4261 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     4321 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     4381 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     4441 ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg
     4501 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     4561 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     4621 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     4681 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     4741 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     4801 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     4861 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     4921 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     4981 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     5041 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     5101 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     5161 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     5221 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     5281 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     5341 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     5401 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     5461 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     5521 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     5581 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     5641 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     5701 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     5761 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     5821 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     5881 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     5941 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6001 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     6061 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     6121 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     6181 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     6241 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     6301 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     6361 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     6421 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     6481 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     6541 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     6601 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa
     6661 gggaacaaaa gctgggtacc gggccccccc tcgaggtcga cggtatcgat aagcttgata
     6721 tcctctgagc tcttaaacag ccatggagca aggaaaacct tgagagaggc ggggttggga
     6781 tatgaggtgc tccatcaaca gcctttagcc ttgcatgcgg tgcagaacta gctggggagg
     6841 actttcaaac tgctgattca caggcctctc accctgtcag tctgtctgtc cctccttcct
     6901 ctggtgggct gccttctact tctgttttgt gtgacgcata gggaagccac ctcacaaagg
     6961 ggaaggagtc gggggttcta cccacctgca gatgatcgtg aatttcaaag actcagatgg
     7021 acaaggcaca tggtgaccca aggcagaata tggaaaagcc agcaagaatc tagagaaggg
     7081 aggaga
//
