LOCUS       sequence-416609-        5865 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminal EGFP-tagging of inserts for inducible expression. Use 
            with Flp-In T-Rex system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5865)
  AUTHORS   Hertz EPT, Vega IA, Kruse T, Wang Y, Hendriks IA, Bizard AH, 
            Eugui-Anta A, Hay RT, Nielsen ML, Nilsson J, Hickson ID, Mailand N
  TITLE     The SUMO-NIP45 pathway processes toxic DNA catenanes to prevent 
            mitotic failure.
  JOURNAL   Nat Struct Mol Biol. 2023 Jul 20. doi: 10.1038/s41594-023-01045-0.
  PUBMED    37474739
REFERENCE   2  (bases 1 to 5865)
  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 Struct 
            Mol Biol. 2023 Jul 20. doi: 10.1038/s41594-023-01045-0."
COMMENT     Sequence Label: pcDNA5-FRT-TO-EGFP-N
FEATURES             Location/Qualifiers
     source          1..5865
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(63..1085)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(63..1085)
                     /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
                     "
     protein_bind    complement(1093..1140)
                     /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)."
     primer_bind     1272..1291
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    1424..1648
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1637..1654
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(join(1766..2476,2477..2479,2480..2482))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1766..2476,2477..2479,2480..2482))
                     /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(1808..1829)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2157..2176
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2416..2437
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2578..2602)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    2612..2630
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2633..2651
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(2653..2856)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2682..2702)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2857..3236
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3408..3427
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3502..3606
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3607..3675,3676..4467)
                     /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(3825..3844)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4638..5226
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5127..5146
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5380..5397
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5514..5535
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5550..5555,5556..5573,5574..5580)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5588..5604
                     /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     5593..5615
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5612..5628
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5612..5628
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5677..5798
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5688..5707)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5742..5761
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 acgattccga agcccaacct ttcatagaag gcggcggtgg aatcgaaatc tcgtagtacg
       61 tgctattcct ttgccctcgg acgagtgctg gggcgtcggt ttccactatc ggcgagtact
      121 tctacacagc catcggtcca gacggccgcg cttctgcggg cgatttgtgt acgcccgaca
      181 gtcccggctc cggatcggac gattgcgtcg catcgaccct gcgcccaagc tgcatcatcg
      241 aaattgccgt caaccaagct ctgatagagt tggtcaagac caatgcggag catatacgcc
      301 cggagccgcg gcgatcctgc aagctccgga tgcctccgct cgaagtagcg cgtctgctgc
      361 tccatacaag ccaaccacgg cctccagaag aagatgttgg cgacctcgta ttgggaatcc
      421 ccgaacatcg cctcgctcca gtcaatgacc gctgttatgc ggccattgtc cgtcaggaca
      481 ttgttggagc cgaaatccgc gtgcacgagg tgccggactt cggggcagtc ctcggcccaa
      541 agcatcagct catcgagagc ctgcgcgacg gacgcactga cggtgtcgtc catcacagtt
      601 tgccagtgat acacatgggg atcagcaatc gcgcatatga aatcacgcca tgtagtgtat
      661 tgaccgattc cttgcggtcc gaatgggccg aacccgctcg tctggctaag atcggccgca
      721 gcgatcgcat ccatggcctc cgcgaccggc tgcagaacag cgggcagttc ggtttcaggc
      781 aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc aataggtcag gctctcgctg
      841 aattccccaa tgtcaagcac ttccggaatc gggagcgcgg ccgatgcaaa gtgccgataa
      901 acataacgat ctttgtagaa accatcggcg cagctattta cccgcaggac atatccacgc
      961 cctcctacat cgaagctgaa agcacgagat tcttcgccct ccgagagctg catcaggtcg
     1021 gagacgctgt cgaacttttc gatcagaaac ttctcgacag acgtcgcggt gagttcaggc
     1081 tttttggcca aggaagttcc tatactttct agagaatagg aacttcggaa taggaacttc
     1141 taggtacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     1201 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     1261 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     1321 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtgg
     1381 ggataccccc tagagcccca gctggttctt tccgcctcag aagccataga gcccaccgca
     1441 tccccagcat gcctgctatt gtcttcccaa tcctccccct tgctgtcctg ccccacccca
     1501 ccccccagaa tagaatgaca cctactcaga caatgcgatg caatttcctc attttattag
     1561 gaaaggacag tgggagtggc accttccagg gtcaaggaag gcacggggga ggggcaaaca
     1621 acagatggct ggcaactaga aggcacagtc gaggctgatc agcgggttta aacgggccct
     1681 ctagactcga gcggccgcca ctgtgctgga tatctgcaga attccaccac actggactag
     1741 tggatccgag ctcggtacca agcttcttgt acagctcgtc catgccgaga gtgatcccgg
     1801 cggcggtcac gaactccagc aggaccatgt gatcgcgctt ctcgttgggg tctttgctca
     1861 gggcggactg ggtgctcagg tagtggttgt cgggcagcag cacggggccg tcgccgatgg
     1921 gggtgttctg ctggtagtgg tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga
     1981 tcttgaagtt caccttgatg ccgttcttct gcttgtcggc catgatatag acgttgtggc
     2041 tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc
     2101 ccttcagctc gatgcggttc accagggtgt cgccctcgaa cttcacctcg gcgcgggtct
     2161 tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg
     2221 cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta gcggctgaag cactgcacgc
     2281 cgtaggtcag ggtggtcacg agggtgggcc agggcacggg cagcttgccg gtggtgcaga
     2341 tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg gacacgctga
     2401 acttgtggcc gtttacgtcg ccgtccagct cgaccaggat gggcaccacc ccggtgaaca
     2461 gctcctcgcc cttgctcacc atggtggaag cttaagttta aacgctagag tccggaggct
     2521 ggatcggtcc cggtgtcttc tatggaggtc aaaacagcgt ggatggcgtc tccaggcgat
     2581 ctgacggttc actaaacgag ctcgtcgacg atctctatca ctgataggga gatctctatc
     2641 actgataggg agagctctgc ttatatagac ctcccaccgt acacgcctac cgcccatttg
     2701 cgtcaatggg gcggagttgt tacgacattt tggaaagtcc cgttgatttt ggtgccaaaa
     2761 caaactccca ttgacgtcaa tggggtggag acttggaaat ccccgtgagt caaaccgcta
     2821 tccacgccca ttgatgtact gccaaaaccg catcaccatg gtaatagcga tgactaatac
     2881 gtagatgtac tgccaagtag gaaagtccca taaggtcatg tactgggcat aatgccaggc
     2941 gggccattta ccgtcattga cgtcaatagg gggcgtactt ggcatatgat acacttgatg
     3001 tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtccc
     3061 tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg ggtcgttggg
     3121 cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg cggaactcca tatatgggct
     3181 atgaactaat gaccccgtaa ttgattacta ttaataacta gtcaataatc aatgtcaacg
     3241 cgtatatctg gcccgtacat cgcgaagcag cgcaaaacgc ctaaccctaa gcagattctt
     3301 catgcaattg tcggtcaagc cttgccttgt tgtagcttaa attttgctcg cgcactactc
     3361 agcgacctcc aacacacaag cagggagcag atactggctt aactatgcgg catcagagca
     3421 gattgtactg agagtgcacc ataggggatc gggagatctc ccgatccgtc gacgtcaggt
     3481 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3541 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     3601 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     3661 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     3721 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     3781 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     3841 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     3901 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     3961 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     4021 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     4081 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     4141 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     4201 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     4261 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     4321 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     4381 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     4441 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     4501 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     4561 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4621 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4681 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4741 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     4801 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4861 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4921 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4981 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     5041 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5101 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5161 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5221 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5281 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     5341 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     5401 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     5461 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     5521 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     5581 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     5641 aagctctagc tagaggtcga cggtatacag acatgataag atacattgat gagtttggac
     5701 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     5761 ctttatttgt aaccattata agctgcaata aacaagttgg ggtgggcgaa gaactccagc
     5821 atgagatccc cgcgctggag gatcatccag ccggcgtccc ggaaa
//
