LOCUS       sequence-217034-        4125 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  General donor vector with T2A-H2B-GFP coding sequence flanking by 
            restriction sites for generating knock-in donor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4125)
  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 4125)
  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: EasyFusion T2A-H2B-GFP
FEATURES             Location/Qualifiers
     source          1..4125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      17..605
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     506..525
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     759..776
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    893..914
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(929..934,935..952,953..959)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    967..983
                     /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     972..994
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     991..1007
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     991..1007
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1026..1046
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1028..1046
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     1075..1091
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1076..1092
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1113..1166
                     /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             1173..1550
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      1563..1572
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1569..1571,1572..1574,1575..2288)
                     /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(1569..1571,1572..1574,1575..2285)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1614..1635)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1875..1894)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2222..2243
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2290..2306)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2290..2306)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2338..2357)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2339..2357)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2367..2384)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2367..2383)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2376..2398)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2524..2979)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2661..2682)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2873..2892
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3006..3110
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3111..3179,3180..3971)
                     /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(3329..3348)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
       61 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      121 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
      181 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      241 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      301 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      361 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      421 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      481 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      541 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      601 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      661 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      721 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      781 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
      841 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
      901 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
      961 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     1021 agcgcgcaat taaccctcac taaagggaac aaaagctggg taccgggccc cccctcgagg
     1081 tcgacggtat cgataagctt gatatcgaat tcgagggcag aggaagtctt ctaacatgcg
     1141 gtgacgtgga ggagaatccc ggccctaccg gtatgccaga gccagcgaag tctgctcccg
     1201 ccccgaaaaa gggctccaag aaggcggtga ctaaggcgca gaagaaaggc ggcaagaagc
     1261 gcaagcgcag ccgcaaggag agctattcca tctatgtgta caaggttctg aagcaggtcc
     1321 accctgacac cggcatttcg tccaaggcca tgggcatcat gaattcgttt gtgaacgaca
     1381 ttttcgagcg catcgcaggt gaggcttccc gcctggcgca ttacaacaag cgctcgacca
     1441 tcacctccag ggagatccag acggccgtgc gcctgctgct gcctggggag ttggccaagc
     1501 acgccgtgtc cgagggtact aaggccatca ccaagtacac cagcgctaag gatccaccgg
     1561 tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     1621 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     1681 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     1741 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     1801 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     1861 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     1921 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     1981 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     2041 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     2101 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     2161 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     2221 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     2281 acaagtaagg atccactagt tctagagcgg ccgccaccgc ggtggagctc caattcgccc
     2341 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
     2401 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     2461 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     2521 tggaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     2581 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     2641 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     2701 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     2761 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     2821 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     2881 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     2941 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa
     3001 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3061 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     3121 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     3181 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     3241 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     3301 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     3361 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     3421 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     3481 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     3541 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     3601 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     3661 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     3721 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     3781 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     3841 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     3901 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     3961 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     4021 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4081 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaa
//
