LOCUS       sequence-213329-        4354 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  General donor vector with T2A-H2B-miRFP703 coding sequence flanking 
            by restriction sites for generating knock-in donor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4354)
  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 4354)
  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-miRFP703
FEATURES             Location/Qualifiers
     source          1..4354
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     complement(16..35)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     226..247
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        410..514
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(515..583,584..1375)
                     /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(733..752)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1546..2134
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2035..2054
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2288..2305
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2422..2443
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2458..2463,2464..2481,2482..2488)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2496..2512
                     /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     2501..2523
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2520..2536
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2520..2536
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2555..2575
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2557..2575
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2604..2620
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2605..2621
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2642..2695
                     /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             2702..3079
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      3092..3101
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3098..4045
                     /codon_start=1
                     /product="bright monomeric near-infrared fluorescent 
                     protein engineered from a bacterial phytochrome, with an 
                     emission maximum of 703 nm (Shcherbakova et al., 2016)"
                     /label=miRFP703
                     /label=ORF frame 1
                     /translation="MVAGHASGSPAFGTASHSNCEHEEIHLAGSIQPHGALLVVSEHDH
                     RVIQASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTE
                     YCGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTG
                     YDRVMVYRFDEQGHGLVFSECHVPGLESYFGNRYPSSLVPQMARQLYVRQRVRVLVDVT
                     YQPVPLEPRLSPLTGRDLDMSGCFLRSMSPIHLQFLKDMGVRATLAVSLVVGGKLWGLV
                     VCHHYLPRFIRFELRAICKRLAERIATRITALES"
     primer_bind     complement(4047..4063)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4047..4063)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4095..4114)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4096..4114)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4124..4141)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4124..4140)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4133..4155)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
       61 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
      121 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
      181 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
      241 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
      301 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
      361 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
      421 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
      481 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
      541 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
      601 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
      661 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
      721 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
      781 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
      841 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
      901 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
      961 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     1021 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     1081 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     1141 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     1201 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     1261 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     1321 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     1381 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     1441 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     1501 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     1561 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     1621 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     1681 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     1741 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     1801 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     1861 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     1921 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     1981 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     2041 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     2101 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     2161 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     2221 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     2281 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     2341 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     2401 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     2461 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     2521 aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca
     2581 aaagctgggt accgggcccc ccctcgaggt cgacggtatc gataagcttg atatcgaatt
     2641 cgagggcaga ggaagtcttc taacatgcgg tgacgtggag gagaatcccg gccctaccgg
     2701 tatgccagag ccagcgaagt ctgctcccgc cccgaaaaag ggctccaaga aggcggtgac
     2761 taaggcgcag aagaaaggcg gcaagaagcg caagcgcagc cgcaaggaga gctattccat
     2821 ctatgtgtac aaggttctga agcaggtcca ccctgacacc ggcatttcgt ccaaggccat
     2881 gggcatcatg aattcgtttg tgaacgacat tttcgagcgc atcgcaggtg aggcttcccg
     2941 cctggcgcat tacaacaagc gctcgaccat cacctccagg gagatccaga cggccgtgcg
     3001 cctgctgctg cctggggagt tggccaagca cgccgtgtcc gagggtacta aggccatcac
     3061 caagtacacc agcgctaagg atccaccggt cgccaccatg gtagcaggtc atgcctctgg
     3121 cagccccgca ttcgggaccg cctctcattc gaattgcgaa catgaagaga tccacctcgc
     3181 cggctcgatc cagccgcatg gcgcgcttct ggtcgtcagc gaacatgatc atcgcgtcat
     3241 ccaggccagc gccaacgccg cggaatttct gaatctcgga agcgtactcg gcgttccgct
     3301 cgccgagatc gacggcgatc tgttgatcaa gatcctgccg catctcgatc ccaccgccga
     3361 aggcatgccg gtcgcggtgc gctgccggat cggcaatccc tctacggagt actgcggtct
     3421 gatgcatcgg cctccggaag gcgggctgat catcgaactc gaacgtgccg gcccgtcgat
     3481 cgatctgtca ggcacgctgg cgccggcgct ggagcggatc cgcacggcgg gttcactgcg
     3541 cgcgctgtgc gatgacaccg tgctgctgtt tcagcagtgc accggctacg accgggtgat
     3601 ggtgtatcgt ttcgatgagc aaggccacgg cctggtattc tccgagtgcc atgtgcctgg
     3661 gctcgaatcc tatttcggca accgctatcc gtcgtcgctg gtcccgcaga tggcgcggca
     3721 gctgtacgtg cggcagcgcg tccgcgtgct ggtcgacgtc acctatcagc cggtgccgct
     3781 ggagccgcgg ctgtcgccgc tgaccgggcg cgatctcgac atgtcgggct gcttcctgcg
     3841 ctcgatgtcg ccgatccatc tgcagttcct gaaggacatg ggcgtgcgcg ccaccctggc
     3901 ggtgtcgctg gtggtcggcg gcaagctgtg gggcctggtt gtctgtcacc attatctgcc
     3961 gcgcttcatc cgtttcgagc tgcgggcgat ctgcaaacgg ctcgccgaaa ggatcgcgac
     4021 gcggatcacc gcgcttgaga gctaaggatc cactagttct agagcggccg ccaccgcggt
     4081 ggagctccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac
     4141 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     4201 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     4261 gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4321 tggttacgcg cagcgtgacc gctacacttg ccag
//
