LOCUS       sequence-251376-        6517 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6517)
  AUTHORS   Meyer C, Garzia A, Morozov P, Molina H, Tuschl T
  TITLE     The G3BP1-Family-USP10 Deubiquitinase Complex Rescues Ubiquitinated 
            40S Subunits of Ribosomes Stalled in Translation from Lysosomal 
            Degradation.
  JOURNAL   Mol Cell. 2020 Mar 19;77(6):1193-1205.e5. doi: 
            10.1016/j.molcel.2019.12.024. Epub 2020 Jan 24.
  PUBMED    31981475
REFERENCE   2  (bases 1 to 6517)
  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: 
            "10.1016/j.molcel.2019.12.024"; journalName: "Mol Cell"; date: 
            "2020-03-19- 19"; volume: "77"; issue: "6"; pages: "1193-1205.e5"
COMMENT     Sequence Label: pcDNA5_FRT_GFP_6TS_mCH
FEATURES             Location/Qualifiers
     source          1..6517
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        140..519
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        520..723
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     674..694
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     768..787
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        768..786
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(845..847,848..850,851..1561)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(845..847,848..850,851..1561)
                     /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(890..911)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1151..1170)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1498..1519
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1616..2326
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1761..1779)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2029..2048
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2374..2391)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2380..2604
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2737..2756)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2888..2935
                     /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             2943..3965
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2943..3965
                     /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    4095..4216
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4132..4151)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4186..4205
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4265..4281)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4265..4281)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4278..4300)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4289..4305
                     /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(4313..4319,4320..4337,4338..4343))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4358..4379
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4496..4513)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4667..5255)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4747..4766)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5426..6217,6218..6286))
                     /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     6049..6068
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6287..6391)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6466..6485)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg
       61 accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
      121 cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg
      181 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
      241 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
      301 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
      361 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
      421 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
      481 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
      541 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
      601 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
      661 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
      721 gctctctggc taactagaga acccactgct tactggctta tcgaaattaa tacgactcac
      781 tatagggaga cccaagctgg ctagcgttta aacttaagct tggtaccgag ctcggatccc
      841 gagtatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
      901 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
      961 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     1021 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     1081 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     1141 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     1201 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1261 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     1321 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1381 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1441 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     1501 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1561 gaagcttact agcactagca ctagcactag cactagcact agcactagca ctagtatggt
     1621 gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca
     1681 catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
     1741 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc
     1801 ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc
     1861 cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat
     1921 gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga
     1981 gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca
     2041 gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct
     2101 gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt
     2161 caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat
     2221 caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc
     2281 cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtagtaag cggccgctcg
     2341 agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc
     2401 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     2461 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     2521 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     2581 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
     2641 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2701 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2761 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2821 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     2881 gtacctagaa gttcctattc cgaagttcct attctctaga aagtatagga acttccttgg
     2941 ccaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca
     3001 gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg
     3061 taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc
     3121 gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg
     3181 gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc
     3241 aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg
     3301 cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa
     3361 tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc
     3421 actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc
     3481 tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct
     3541 ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga
     3601 tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt
     3661 gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc
     3721 ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg
     3781 gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag
     3841 ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct
     3901 gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg
     3961 aatagcacgt actacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     4021 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     4081 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     4141 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     4201 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     4261 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4321 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4381 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4441 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4501 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4561 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4621 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4681 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4741 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4801 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4861 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4921 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4981 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5041 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5101 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5161 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5221 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5281 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5341 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5401 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5461 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5521 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5581 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5641 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5701 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5761 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5821 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5881 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5941 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6001 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6061 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6121 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6181 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6241 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6301 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6361 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     6421 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
     6481 tgccgcatag ttaagccagt atctgctccc tgcttgt
//
