LOCUS       sequence-273459-        7775 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses eGFP-tagged HROB in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7775)
  AUTHORS   Hustedt N, Saito Y, Zimmermann M, Alvarez-Quilon A, Setiaputra D, 
            Adam S, McEwan A, Yuan JY, Olivieri M, Zhao Y, Kanemaki MT, 
            Jurisicova A, Durocher D
  TITLE     Control of homologous recombination by the HROB-MCM8-MCM9 pathway.
  JOURNAL   Genes Dev. 2019 Oct 1;33(19-20):1397-1415. doi: 
            10.1101/gad.329508.119. Epub 2019 Aug 29.
  PUBMED    31467087
REFERENCE   2  (bases 1 to 7775)
  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: "Genes 
            Dev."; date: "2019-10-1"; volume: "33(19-20):1397-1415. doi"; pages:
            " 10.1101/gad.329508.119. Epub 2019 Aug 29"
COMMENT     Sequence Label: pcDNA5-FRT-T0-eGFP-HROB
FEATURES             Location/Qualifiers
     source          1..7775
                     /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"
     primer_bind     complement(3113..3130)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3119..3343
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3476..3495)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3627..3674
                     /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             3682..4704
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4834..4955
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4871..4890)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4925..4944
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5004..5020)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5004..5020)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5017..5039)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5028..5044
                     /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(5052..5058,5059..5076,5077..5082))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5097..5118
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5235..5252)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5406..5994)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5486..5505)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6165..6956,6957..7025))
                     /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     6788..6807
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7026..7130)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7205..7224)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7396..7775
                     /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 agcgtgcagt ttgcagaagc tgtttgctgt ggaagaggag
     1141 tttgaagatg aggatttctt gtctgctgtg gaggatgcag agaaccggtt tactggctca
     1201 ctgcctgtga atgctgggcg cctgagacct gtctcttcta ggccacagga gactgtgcag
     1261 gcacagtcct ccaggctgct gctgttacac cccactgctc cctcagaggc tttgggcctg
     1321 ccagacttgg acctctgcct ccctgcctcc agcacgccca gtgctgacag ccgtccatca
     1381 tgcataggag cagctcccct aaggcctgtc tctacttcca gcagctggat tggcaatcag
     1441 agaagagtga cagtgacaga agtgctcaga gagacagcaa gacctcagtc ctcagcctta
     1501 caccccctac tcacctttga gagccaacag cagcaagttg gtggctttga ggggcctgaa
     1561 caagacgaat ttgataaagt cctggcaagc atggagttgg aggagcctgg catggagctg
     1621 gaatgtggag tcagcagtga ggccatacca atcctgcctg cccagcagcg ggagggttca
     1681 gtattggcta aaaaagcccg ggtagttgat ctgagtggat cttgccagaa ggggcctgtg
     1741 cctgccatcc acaaagcggg tatcatgtcc gcccaggatg agtctctaga tcctgtcatc
     1801 caatgtagga ctccacgacc ccccttgaga cctggtgctg tgggtcacct tcctgttcca
     1861 actgccttaa cagttcccac tcagcaactc cactgggaag tctgtccgca acgctcccct
     1921 gttcaagcac ttcagcctct ccaagctgct agagggacca ttcagagcag ccctcaaaat
     1981 cgtttccctt gtcagccatt ccagtctcca agttcctggt taagtggcaa agctcattta
     2041 cccagacctc gaactcccaa ctcaagctgt tctactccct caaggactag ctctggatta
     2101 tttcctcgga tacccttaca accgcaagct ccagtgtctt ccattgggtc tcctgttggt
     2161 accccaaaag gtccccaggg agctctgcag acacccatag tcaccaacca cctggtgcag
     2221 ctagtcactg ctgccagccg gacaccccag cagcccaccc atccctccac ccgagccaaa
     2281 actcgccgtt tccctggccc agctgggatc ctgcctcacc agcagagtgg gagaagtctg
     2341 gaggacatca tggtttccgc gccccaaact ccaacccatg gtgctctggc taaattccag
     2401 acagagattg ttgctagttc ccaggcatct gtggaggagg attttgggcg agggccctgg
     2461 ctgaccatga aatccacgct aggcctggat gagagagacc ctagctgctt cctctgtacc
     2521 tacagcattg tcatggtgct gcgcaagcag gcagccctga agcagcttcc taggaacaag
     2581 gtccccaaca tggcggtgat gatcaagtcc ctgactcgga gcacaatgga cgccagtgtg
     2641 gttttcaagg accccacggg agagatgcag gggacggtgc acaggttgct gctggagacg
     2701 tgccagaatg agctgaagcc tggctcagtg ctgctgctga agcagattgg agtgttttct
     2761 ccttcacttc gaaatcacta cctcaacgtg acacccaaca acctggtcca tatttacagc
     2821 ccggattctg gggatgggag cttcctcaag ccatctcagc ccttccccaa ggattcaggg
     2881 agcttccagc atgatgtggc tgcaaagccc gaggaaggct tcagaacagc acagaaccta
     2941 gaggcagagg cgtcccctga ggaagaactc ccagaagcag atgacctgga tggactcctg
     3001 agtgagcttc ctgaagactt cttctgtggg accagtagtt gagaattctg cagatatcca
     3061 tcacactggc ggccgctcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact
     3121 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     3181 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     3241 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     3301 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     3361 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     3421 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     3481 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     3541 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3601 gattagggtg atggttcacg tacctagaag ttcctattcc gaagttccta ttctctagaa
     3661 agtataggaa cttccttggc caaaaagcct gaactcaccg cgacgtctgt cgagaagttt
     3721 ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct
     3781 cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc
     3841 gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt
     3901 ccggaagtgc ttgacattgg ggagttcagc gagagcctga cctattgcat ctcccgccgt
     3961 gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg
     4021 gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc
     4081 ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt
     4141 gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc
     4201 gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc
     4261 gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc
     4321 attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc
     4381 tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg
     4441 gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc
     4501 tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac
     4561 gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg
     4621 gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc
     4681 actcgtccga gggcaaagga atagcacgta ctacgagatt tcgattccac cgccgccttc
     4741 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4801 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     4861 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4921 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     4981 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     5041 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     5101 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5161 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5221 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5281 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5341 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5401 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5461 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5521 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5581 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5641 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5701 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5761 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5821 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     5881 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5941 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6001 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6061 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6121 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6181 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6241 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6301 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6361 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6421 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6481 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6541 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6601 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6661 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6721 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6781 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6841 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6901 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6961 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7021 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     7081 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     7141 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc
     7201 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
     7261 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
     7321 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
     7381 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
     7441 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     7501 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     7561 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     7621 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     7681 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     7741 agtacatcta cgtattagtc atcgctatta ccatg
//
