LOCUS       sequence-218413-        6497 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminally tagged eGFP-REV7 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6497)
  AUTHORS   Noordermeer SM, Adam S, Setiaputra D, Barazas M, Pettitt SJ, Ling 
            AK, Olivieri M, Alvarez-Quilon A, Moatti N, Zimmermann M, Annunziato
            S, Krastev DB, Song F, Brandsma I, Frankum J, Brough R, Sherker A, 
            Landry S, Szilard RK, Munro MM, McEwan A, Goullet de Rugy T, Lin ZY,
            Hart T, Moffat J, Gingras AC, Martin A, van Attikum H, Jonkers J, 
            Lord CJ, Rottenberg S, Durocher D
  TITLE     The shieldin complex mediates 53BP1-dependent DNA repair.
  JOURNAL   Nature. 2018 Aug;560(7716):117-121. doi: 10.1038/s41586-018-0340-7. 
            Epub 2018 Jul 18.
  PUBMED    30022168
REFERENCE   2  (bases 1 to 6497)
  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"; doi: 
            "10.1038/s41586-018-0340-7"; journalName: "Nature"; date: "2018-08";
            volume: "560"; issue: "7716"; pages: "117-121"
COMMENT     Sequence Label: pDEST-FRT/TO-eGFP-REV7
FEATURES             Location/Qualifiers
     source          1..6497
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    206..224
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    227..245
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     255..279
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(376..378,379..381,382..1092)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(376..378,379..381,382..1092)
                     /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(421..442)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(682..701)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1029..1050
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1102..1126
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1769..1793)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1835..1852)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1841..2065
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2198..2217)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2349..2396
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2404..3426
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3556..3677
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3593..3612)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3647..3666
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3726..3742)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3726..3742)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3739..3761)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3750..3766
                     /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)."
     promoter        complement(join(3774..3780,3781..3798,3799..3804))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3819..3840
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3957..3974)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4128..4716)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4208..4227)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4887..5678,5679..5747))
                     /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     5510..5529
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5748..5852)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5927..5946)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6118..6497
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctctccct atcagtgata gagatctccc tatcagtgat
      241 agagatcgtc gacgagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc
      301 tgttttgacc tccatagaag acaccgggac cgatccagcc tccggactct agcgtttaaa
      361 cttaagcttg gtaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
      421 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
      481 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
      541 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
      601 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
      661 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
      721 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
      781 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
      841 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
      901 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
      961 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     1021 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     1081 gagctgtaca agggcgcgcc cacaagtttg tacaaaaaag caggctccac catgaccacg
     1141 ctcacacgac aagacctcaa ctttggccaa gtggtggccg atgtgctctg cgagttcctg
     1201 gaggtggctg tgcatctcat cctctacgtg cgcgaggtct accccgtggg catcttccag
     1261 aaacgcaaga agtacaacgt gccggtccag atgtcctgcc acccggagct gaatcagtat
     1321 atccaggaca cgctgcactg cgtcaagcca ctcctggaga agaatgatgt ggagaaagtg
     1381 gtggtggtga ttttggataa agagcaccgc ccagtggaga aattcgtctt tgagatcacc
     1441 cagcctccac tgctgtccat cagctcagac tcgctgttgt ctcatgtgga gcagctgctc
     1501 cgggccttca tcctgaagat cagcgtgtgc gatgccgtcc tggaccacaa ccccccaggc
     1561 tgtaccttca cagtcctggt gcacacgaga gaagccgcca ctcgcaacat ggagaagatc
     1621 caggtcatca aggatttccc ctggatcctg gcggatgagc aggatgtcca catgcatgac
     1681 ccccggctga taccactaaa aaccatgacg tcggacattt taaagatgca gctttacgtg
     1741 gaagagcgcg ctcataaagg cagctaggac ccagctttct tgtacaaagt ggtgtgactc
     1801 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     1861 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     1921 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     1981 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     2041 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     2101 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     2161 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     2221 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     2281 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     2341 cgtacctaga agttcctatt ccgaagttcc tattctctag aaagtatagg aacttccttg
     2401 gccaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac
     2461 agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat
     2521 gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat
     2581 cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt
     2641 ggggagttca gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg
     2701 caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat
     2761 gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga
     2821 atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat
     2881 cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag
     2941 ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc
     3001 tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg
     3061 atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct
     3121 tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg
     3181 cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac
     3241 ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga
     3301 gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc
     3361 tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag
     3421 gaatagcacg tactacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     3481 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     3541 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     3601 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     3661 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     3721 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     3781 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     3841 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3901 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     3961 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4021 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4081 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4141 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4201 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4261 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4321 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4381 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4441 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4501 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4561 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4621 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4681 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4741 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4801 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4861 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4921 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4981 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5041 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5101 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5161 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5221 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5281 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5341 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5401 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5461 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5521 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5581 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5641 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     5701 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     5761 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5821 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     5881 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     5941 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     6001 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
     6061 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
     6121 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
     6181 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     6241 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     6301 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     6361 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     6421 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     6481 tcatcgctat taccatg
//
