LOCUS       sequence-174035-        7070 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 7070)
  AUTHORS   Fan W, Tang Z, Chen D, Moughon D, Ding X, Chen S, Zhu M, Zhong Q
  TITLE     Keap1 facilitates p62-mediated ubiquitin aggregate clearance via 
            autophagy.
  JOURNAL   Autophagy. 2010 Jul 22. 6(5):.
  PUBMED    20495340
REFERENCE   2  (bases 1 to 7070)
  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: "Autophagy. 
            2010 Jul 22. 6(5):."
COMMENT     Sequence Label: Flag-Keap1
FEATURES             Location/Qualifiers
     source          1..7070
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        37..416
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        417..620
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     571..591
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    622..640
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    643..661
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     671..695
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2718..2783
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2824..2841)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2830..3054
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3187..3206)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3338..3385
                     /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             3393..4415
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3393..4415
                     /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    4545..4666
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4582..4601)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4636..4655
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4715..4731)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4715..4731)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4728..4750)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4739..4755
                     /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(4763..4769,4770..4787,4788..4793))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4808..4829
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4946..4963)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5117..5705)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5197..5216)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5876..6667,6668..6736))
                     /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     6499..6518
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6737..6841)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6916..6935)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt
       61 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      121 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      181 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      241 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      301 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      361 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
      421 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
      481 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
      541 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
      601 aggtctatat aagcagagct ctccctatca gtgatagaga tctccctatc agtgatagag
      661 atcgtcgacg agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt
      721 ttgacctcca tagaagacac cgggaccgat ccagcctccg gactctagcg tttaaactta
      781 agcttggtac cgagctcgga tccactagtc cagtgtggtg gaattctgca gatatcatgc
      841 agccagatcc caggcctagc ggggctgggg cctgctgccg attcctgccc ctgcagtcac
      901 agtgccctga gggggcaggg gacgcggtga tgtacgcctc cactgagtgc aaggcggagg
      961 tgacgccctc ccagcatggc aaccgcacct tcagctacac cctggaggat cataccaagc
     1021 aggcctttgg catcatgaac gagctgcggc tcagccagca gctgtgtgac gtcacactgc
     1081 aggtcaagta ccaggatgca ccggccgccc agttcatggc ccacaaggtg gtgctggcct
     1141 catccagccc tgtcttcaag gccatgttca ccaacgggct gcgggagcag ggcatggagg
     1201 tggtgtccat tgagggtatc caccccaagg tcatggagcg cctcattgaa ttcgcctaca
     1261 cggcctccat ctccatgggc gagaagtgtg tcctccacgt catgaacggt gctgtcatgt
     1321 accagatcga cagcgttgtc cgtgcctgca gtgacttcct ggtgcagcag ctggacccca
     1381 gcaatgccat cggcatcgcc aacttcgctg agcagattgg ctgtgtggag ttgcaccagc
     1441 gtgcccggga gtacatctac atgcattttg gggaggtggc caagcaagag gagttcttca
     1501 acctgtccca ctgccaactg gtgaccctca tcagccggga cgacctgaac gtgcgctgcg
     1561 agtccgaggt cttccacgcc tgcatcaact gggtcaagta cgactgcgaa cagcgacggt
     1621 tctacgtcca ggcgctgctg cgggccgtgc gctgccactc gttgacgccg aacttcctgc
     1681 agatgcagct gcagaagtgc gagatcctgc agtccgactc ccgctgcaag gactacctgg
     1741 tcaagatctt cgaggagctc accctgcaca agcccacgca ggtgatgccc tgccgggcgc
     1801 ccaaggtggg ccgcctgatc tacaccgcgg gcggctactt ccgacagtcg ctcagctacc
     1861 tggaggctta caaccccagt gacggcacct ggctccggtt ggcggacctg caggtgccgc
     1921 ggagcggcct ggccggctgc gtggtgggcg ggctgttgta cgccgtgggc ggcaggaaca
     1981 actcgcccga cggcaacacc gactccagcg ccctggactg ttacaacccc atgaccaatc
     2041 agtggtcgcc ctgcgccccc atgagcgtgc cccgtaaccg catcggggtg ggggtcatcg
     2101 atggccacat ctatgccgtc ggcggctccc acggctgcat ccaccacaac agtgtggaga
     2161 ggtatgagcc agagcgggat gagtggcact tggtggcccc aatgctgaca cgaaggatcg
     2221 gggtgggcgt ggctgtcctc aatcgtctcc tttatgccgt ggggggcttt gacgggacaa
     2281 accgccttaa ttcagctgag tgttactacc cagagaggaa cgagtggcga atgatcacag
     2341 caatgaacac catccgaagc ggggcaggcg tctgcgtcct gcacaactgt atctatgctg
     2401 ctgggggcta tgatggtcag gaccagctga acagcgtgga gcgctacgat gtggaaacag
     2461 agacgtggac tttcgtagcc cccatgaagc accggcgaag tgccctgggg atcactgtcc
     2521 accaggggag aatctacgtc cttggaggct atgatggtca cacgttcctg gacagtgtgg
     2581 agtgttacga cccagataca gacacctgga gcgaggtgac ccgaatgaca tcgggccgga
     2641 gtggggtggg cgtggctgtc accatggagc cctgccggaa gcagattgac cagcagaact
     2701 gtacctgtgg cggccgcgac tacaaagacc atgacggtga ttataaagat catgacatcg
     2761 actacaagga tgacgatgac aagtgactcg agtctagagg gcccgtttaa acccgctgat
     2821 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     2881 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     2941 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     3001 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     3061 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     3121 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3181 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3241 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3301 ccaaaaaact tgattagggt gatggttcac gtacctagaa gttcctattc cgaagttcct
     3361 attctctaga aagtatagga acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg
     3421 tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg
     3481 gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa
     3541 atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg
     3601 cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca
     3661 tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg
     3721 ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga
     3781 gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca
     3841 tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca
     3901 gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag
     3961 tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca
     4021 taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca
     4081 acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc
     4141 ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc
     4201 ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg
     4261 gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc
     4321 gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc
     4381 gacgccccag cactcgtccg agggcaaagg aatagcacgt actacgagat ttcgattcca
     4441 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4501 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4561 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4621 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     4681 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4741 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4801 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4861 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4921 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4981 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5041 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5101 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5161 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5221 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5281 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5341 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5401 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5461 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5521 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5581 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5641 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5701 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5761 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5821 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5881 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5941 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6001 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6061 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6121 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6181 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6241 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6301 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6361 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6421 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6481 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6541 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6601 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6661 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6721 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6781 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6841 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     6901 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     6961 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
     7021 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
//
