LOCUS       sequence-217026-        3672 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  General donor vector with EGFP 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 3672)
  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 3672)
  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 eGFP
FEATURES             Location/Qualifiers
     source          1..3672
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      15..603
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     504..523
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     757..774
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    891..912
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(927..932,933..950,951..957)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    965..981
                     /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     970..992
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     989..1005
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     989..1005
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1024..1044
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1026..1044
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     1073..1089
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1074..1090
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(1111..1113,1114..1116,1117..1827)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1111..1113,1114..1116,1117..1827)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1156..1177)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1417..1436)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1764..1785
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1835..1851)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1835..1851)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1883..1902)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1884..1902)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1912..1929)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1912..1928)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1921..1943)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2069..2524)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2206..2227)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2418..2437
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2551..2655
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2656..2724,2725..3516)
                     /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(2874..2893)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
       61 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
      121 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
      181 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
      241 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
      301 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
      361 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
      421 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
      481 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
      541 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
      601 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
      661 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
      721 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
      781 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
      841 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
      901 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
      961 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
     1021 cgcgcaatta accctcacta aagggaacaa aagctgggta ccgggccccc cctcgaggtc
     1081 gacggtatcg ataagcttga tatcgaattc atggtgagca agggcgagga gctgttcacc
     1141 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     1201 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     1261 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     1321 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     1381 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     1441 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     1501 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     1561 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     1621 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     1681 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     1741 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     1801 actctcggca tggacgagct gtacaagccc gggggatcca ctagttctag agcggccgcc
     1861 accgcggtgg agctccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt
     1921 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     1981 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     2041 acagttgcgc agcctgaatg gcgaatggaa attgtaagcg ttaatatttt gttaaaattc
     2101 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     2161 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     2221 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     2281 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     2341 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     2401 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     2461 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     2521 gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2581 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2641 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     2701 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     2761 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     2821 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     2881 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     2941 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     3001 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     3061 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     3121 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     3181 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     3241 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     3301 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     3361 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     3421 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3481 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     3541 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     3601 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3661 cccgtagaaa ag
//
