LOCUS       sequence-430524-        6727 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a His6-Ubiquitin-Clu(228-238)-eGFP-Clu(204-227) fusion
            protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6727)
  TITLE     Hartl Lab plasmids
REFERENCE   2  (bases 1 to 6727)
  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"
COMMENT     Sequence Label: pHUE-eGFP-Clu-tails
FEATURES             Location/Qualifiers
     source          1..6727
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     806..825
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(845..922)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1128..1147
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1231..1250
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1231..1249
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1250..1274
                     /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)."
     RBS             1289..1311
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1330..1347
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1357..1374
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1378..1605
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             join(1639..1641,1642..1644,1645..2355)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1639..1641,1642..1644,1645..2355)
                     /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(1684..1705)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1945..1964)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2292..2313
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2485..2503)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2499..2546
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2749..2767)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2835..2939
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2940..3008,3009..3800)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3158..3177)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3971..4559
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4460..4479
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4713..4730
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4745..4884
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4810..4829)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4970..4992
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4986..5177)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6489..6727,1..844))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
       61 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
      121 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
      181 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
      241 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
      301 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
      361 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
      421 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
      481 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
      541 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
      601 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
      661 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
      721 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
      781 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
      841 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
      901 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
      961 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     1021 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     1081 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     1141 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     1201 tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag
     1261 cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg
     1321 ggcagcagcc atcatcatca tcatcacagc agcggcctgg tgccgcgcgg cagccatatg
     1381 cagatctttg tgaagaccct cactggcaaa accatcaccc ttgaggtcga gcccagtgac
     1441 accattgaga atgtcaaagc caaaattcaa gacaaggagg gtatcccacc tgaccagcag
     1501 cgtctgatat ttgccggcaa acagctggag gatggccgca ctctctcaga ctacaacatc
     1561 cagaaagagt ccaccctgca cctggtgttg cgcctccgcg gtggtagctt gatgcccttc
     1621 tctccgtacg agcccctgat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     1681 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     1741 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     1801 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     1861 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     1921 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     1981 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     2041 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     2101 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     2161 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     2221 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     2281 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     2341 gacgagctgt acaagtacca ctacctgccc ttcagcctgc cccaccggag gcctcacttc
     2401 ttctttccca agtcccgcat cgtccgctaa gcttagatcc ggctgctaac aaagcccgaa
     2461 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     2521 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga tatcccgcaa
     2581 gaggcccggc agtaccggca taaccaagcc tatgcctaca gcatccaggg tgacggtgcc
     2641 gaggatgacg atgagcgcat tgttagattt catacacggt gcctgactgc gttagcaatt
     2701 taactgtgat aaactaccgc attaaagcta attcttgaag acgaaagggc ctcgtgatac
     2761 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     2821 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2881 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     2941 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     3001 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     3061 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     3121 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     3181 gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     3241 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     3301 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     3361 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     3421 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     3481 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     3541 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     3601 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     3661 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     3721 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     3781 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     3841 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     3901 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     3961 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     4021 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     4081 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     4141 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     4201 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     4261 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     4321 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     4381 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     4441 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     4501 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     4561 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     4621 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     4681 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     4741 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     4801 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     4861 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     4921 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4981 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     5041 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     5101 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     5161 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     5221 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     5281 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     5341 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     5401 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     5461 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     5521 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     5581 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     5641 cgtggccagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga gatggcggac
     5701 gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga
     5761 ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct tccattcagg
     5821 tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg
     5881 cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcggca taaatcgccg
     5941 tgacgatcag cggtccaatg atcgaagtta ggctggtaag agccgcgagc gatccttgaa
     6001 gctgtccctg atggtcgtca tctacctgcc tggacagcat ggcctgcaac gcgggcatcc
     6061 cgatgccgcc ggaagcgaga agaatcataa tggggaaggc catccagcct cgcgtcgcga
     6121 acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct
     6181 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
     6241 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
     6301 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
     6361 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     6421 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     6481 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     6541 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     6601 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     6661 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     6721 gggatat
//
