LOCUS       sequence-215844-        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"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(990..992,993..995,996..1706)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(990..992,993..995,996..1706)
                     /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(1035..1056)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1296..1315)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1643..1664
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1716..1740
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2383..2407)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2449..2466)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2455..2679
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2812..2831)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2963..3010
                     /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             3018..4040
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3018..4040
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4170..4291
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4207..4226)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4261..4280
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4340..4356)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4340..4356)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4353..4375)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4364..4380
                     /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(4388..4394,4395..4412,4413..4418))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4433..4454
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4571..4588)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4742..5330)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4822..4841)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5501..6292,6293..6361))
                     /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     6124..6143
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6362..6466)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttggtacca tggtgagcaa gggcgaggag ctgttcaccg
     1021 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     1081 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     1141 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     1201 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     1261 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     1321 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     1381 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     1441 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     1501 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     1561 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     1621 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     1681 ctctcggcat ggacgagctg tacaagggcg cgcccacaag tttgtacaaa aaagcaggct
     1741 ccaccatgac cacgctcaca cgacaagacc tcaactttgg ccaagtggtg gccgatgtgc
     1801 tctgcgagtt cctggaggtg gctgtgcatc tcatcctcta cgtgcgcgag gtctaccccg
     1861 tgggcatctt ccagaaacgc aagaagtaca acgtgccggt ccagatgtcc tgccacccgg
     1921 agctgaatca gtatatccag gacacgctgc actgcgtcaa gccactcctg gagaagaatg
     1981 atgtggagaa agtggtggtg gtgattttgg ataaagagca ccgcccagtg gagaaattcg
     2041 tctttgagat cacccagcct ccactgctgt ccatcagctc agactcgctg ttgtctcatg
     2101 tggagcagct gctccgggcc ttcatcctga agatcagcgt gtgcgatgcc gtcctggacc
     2161 acaacccccc aggctgtacc ttcacagtcc tggtgcacac gagagaagcc gccactcgca
     2221 acatggagaa gatccaggtc atcaaggatt tcccctggat cctggcggat gagcaggatg
     2281 tccacatgca tgacccccgg ctgataccac taaaaaccat gacgtcggac attttaaaga
     2341 tgcagcttta cgtggaagag cgcgctcata aaggcagcta ggacccagct ttcttgtaca
     2401 aagtggtgtg actcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc
     2461 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     2521 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     2581 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     2641 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     2701 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2761 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2821 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2881 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2941 agggtgatgg ttcacgtacc tagaagttcc tattccgaag ttcctattct ctagaaagta
     3001 taggaacttc cttggccaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga
     3061 tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg
     3121 ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg
     3181 gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg
     3241 aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac
     3301 agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg
     3361 cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat
     3421 tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg
     3481 atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc
     3541 aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc
     3601 acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg
     3661 actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga
     3721 ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc
     3781 ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc
     3841 agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa
     3901 tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg
     3961 tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc
     4021 gtccgagggc aaaggaatag cacgtactac gagatttcga ttccaccgcc gccttctatg
     4081 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     4141 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     4201 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4261 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     4321 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4381 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4441 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4501 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4561 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4621 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4681 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4741 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4801 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4861 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4921 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4981 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5041 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5101 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5161 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5221 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5281 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5341 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5401 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5461 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5521 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5581 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5641 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5701 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5761 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5821 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5881 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5941 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6001 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6061 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6121 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6181 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6241 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6301 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6361 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6421 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6481 aagtgccacc tgacgtc
//
