LOCUS       sequence-217816-        4353 bp    DNA     circular SYN 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      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4353)
  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 4353)
  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..4353
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     107..124
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    241..262
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(277..282,283..300,301..307)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    315..331
                     /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     320..342
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     339..355
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     339..355
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     374..394
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        376..394
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     423..439
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     424..440
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             461..514
                     /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             521..898
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      911..920
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             917..1864
                     /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(1866..1882)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1866..1882)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1914..1933)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1915..1933)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1943..1960)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1943..1959)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1952..1974)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2100..2555)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2237..2258)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2449..2468
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2582..2686
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2687..2755,2756..3547)
                     /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(2905..2924)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3718..4306
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4207..4226
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
       61 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
      121 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
      181 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
      241 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
      301 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      361 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctgggta ccgggccccc
      421 cctcgaggtc gacggtatcg ataagcttga tatcgaattc gagggcagag gaagtcttct
      481 aacatgcggt gacgtggagg agaatcccgg ccctaccggt atgccagagc cagcgaagtc
      541 tgctcccgcc ccgaaaaagg gctccaagaa ggcggtgact aaggcgcaga agaaaggcgg
      601 caagaagcgc aagcgcagcc gcaaggagag ctattccatc tatgtgtaca aggttctgaa
      661 gcaggtccac cctgacaccg gcatttcgtc caaggccatg ggcatcatga attcgtttgt
      721 gaacgacatt ttcgagcgca tcgcaggtga ggcttcccgc ctggcgcatt acaacaagcg
      781 ctcgaccatc acctccaggg agatccagac ggccgtgcgc ctgctgctgc ctggggagtt
      841 ggccaagcac gccgtgtccg agggtactaa ggccatcacc aagtacacca gcgctaagga
      901 tccaccggtc gccaccatgg tagcaggtca tgcctctggc agccccgcat tcgggaccgc
      961 ctctcattcg aattgcgaac atgaagagat ccacctcgcc ggctcgatcc agccgcatgg
     1021 cgcgcttctg gtcgtcagcg aacatgatca tcgcgtcatc caggccagcg ccaacgccgc
     1081 ggaatttctg aatctcggaa gcgtactcgg cgttccgctc gccgagatcg acggcgatct
     1141 gttgatcaag atcctgccgc atctcgatcc caccgccgaa ggcatgccgg tcgcggtgcg
     1201 ctgccggatc ggcaatccct ctacggagta ctgcggtctg atgcatcggc ctccggaagg
     1261 cgggctgatc atcgaactcg aacgtgccgg cccgtcgatc gatctgtcag gcacgctggc
     1321 gccggcgctg gagcggatcc gcacggcggg ttcactgcgc gcgctgtgcg atgacaccgt
     1381 gctgctgttt cagcagtgca ccggctacga ccgggtgatg gtgtatcgtt tcgatgagca
     1441 aggccacggc ctggtattct ccgagtgcca tgtgcctggg ctcgaatcct atttcggcaa
     1501 ccgctatccg tcgtcgctgg tcccgcagat ggcgcggcag ctgtacgtgc ggcagcgcgt
     1561 ccgcgtgctg gtcgacgtca cctatcagcc ggtgccgctg gagccgcggc tgtcgccgct
     1621 gaccgggcgc gatctcgaca tgtcgggctg cttcctgcgc tcgatgtcgc cgatccatct
     1681 gcagttcctg aaggacatgg gcgtgcgcgc caccctggcg gtgtcgctgg tggtcggcgg
     1741 caagctgtgg ggcctggttg tctgtcacca ttatctgccg cgcttcatcc gtttcgagct
     1801 gcgggcgatc tgcaaacggc tcgccgaaag gatcgcgacg cggatcaccg cgcttgagag
     1861 ctaaggatcc actagttcta gagcggccgc caccgcggtg gagctccaat tcgccctata
     1921 gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     1981 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     2041 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga
     2101 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     2161 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     2221 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     2281 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     2341 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     2401 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     2461 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     2521 acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt
     2581 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     2641 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     2701 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     2761 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     2821 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     2881 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     2941 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     3001 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     3061 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     3121 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     3181 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     3241 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     3301 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     3361 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     3421 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     3481 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     3541 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     3601 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     3661 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     3721 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     3781 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     3841 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     3901 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     3961 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     4021 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     4081 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     4141 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     4201 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     4261 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     4321 ggccttttta cggttcctgg ccttttgctg gcc
//
