LOCUS       sequence-217815-        4116 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  General donor vector with T2A-H2B-mCherry 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 4116)
  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 4116)
  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-mCherry
FEATURES             Location/Qualifiers
     source          1..4116
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     142..159
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    276..297
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(312..317,318..335,336..342)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    350..366
                     /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     355..377
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     374..390
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     374..390
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     409..429
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        411..429
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     458..474
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     459..475
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             496..549
                     /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             556..933
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      946..955
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             952..1662
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1097..1115)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1365..1384
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1664..1680)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1664..1680)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1712..1731)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1713..1731)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1741..1758)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1741..1757)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1750..1772)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(1898..2353)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2035..2056)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2247..2266
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2380..2484
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2485..2553,2554..3345)
                     /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(2703..2722)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3516..4104
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4005..4024
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
       61 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
      121 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
      181 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
      241 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
      301 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
      361 taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct
      421 cactaaaggg aacaaaagct gggtaccggg ccccccctcg aggtcgacgg tatcgataag
      481 cttgatatcg aattcgaggg cagaggaagt cttctaacat gcggtgacgt ggaggagaat
      541 cccggcccta ccggtatgcc agagccagcg aagtctgctc ccgccccgaa aaagggctcc
      601 aagaaggcgg tgactaaggc gcagaagaaa ggcggcaaga agcgcaagcg cagccgcaag
      661 gagagctatt ccatctatgt gtacaaggtt ctgaagcagg tccaccctga caccggcatt
      721 tcgtccaagg ccatgggcat catgaattcg tttgtgaacg acattttcga gcgcatcgca
      781 ggtgaggctt cccgcctggc gcattacaac aagcgctcga ccatcacctc cagggagatc
      841 cagacggccg tgcgcctgct gctgcctggg gagttggcca agcacgccgt gtccgagggt
      901 actaaggcca tcaccaagta caccagcgct aaggatccac cggtcgccac catggtgagc
      961 aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg
     1021 gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
     1081 gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg
     1141 gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac
     1201 atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac
     1261 ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc
     1321 atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag
     1381 aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag
     1441 ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag
     1501 accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag
     1561 ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag
     1621 ggccgccact ccaccggcgg catggacgag ctgtacaagt aaggatccac tagttctaga
     1681 gcggccgcca ccgcggtgga gctccaattc gccctatagt gagtcgtatt acgcgcgctc
     1741 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     1801 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     1861 cccttcccaa cagttgcgca gcctgaatgg cgaatggaaa ttgtaagcgt taatattttg
     1921 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     1981 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     2041 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     2101 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     2161 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     2221 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     2281 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     2341 ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     2401 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     2461 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     2521 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     2581 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     2641 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     2701 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     2761 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     2821 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     2881 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     2941 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     3001 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     3061 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     3121 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     3181 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     3241 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     3301 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     3361 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     3421 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     3481 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     3541 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3601 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3661 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3721 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3781 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3841 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3901 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3961 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     4021 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     4081 tcaggggggc ggagcctatg gaaaaacgcc agcaac
//
