LOCUS       sequence-476189-        6694 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a His6-Ubiquitin-eGFP-Clu(204-227) fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6694)
  TITLE     Hartl Lab plasmids 2025
REFERENCE   2  (bases 1 to 6694)
  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-beta-tail
FEATURES             Location/Qualifiers
     source          1..6694
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    40..64
                     /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             79..101
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             120..137
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             147..164
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             168..395
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             join(396..398,399..401,402..1112)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(396..398,399..401,402..1112)
                     /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(441..462)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(702..721)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1049..1070
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1242..1260)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1256..1303
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1506..1524)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1592..1696
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1697..1765,1766..2557)
                     /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(1915..1934)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2728..3316
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3217..3236
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3470..3487
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3502..3641
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3567..3586)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3727..3749
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3743..3934)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5246..6328)
                     /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"
     primer_bind     6290..6309
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6329..6406)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6612..6631
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
       61 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggcagcagcc
      121 atcatcatca tcatcacagc agcggcctgg tgccgcgcgg cagccatatg cagatctttg
      181 tgaagaccct cactggcaaa accatcaccc ttgaggtcga gcccagtgac accattgaga
      241 atgtcaaagc caaaattcaa gacaaggagg gtatcccacc tgaccagcag cgtctgatat
      301 ttgccggcaa acagctggag gatggccgca ctctctcaga ctacaacatc cagaaagagt
      361 ccaccctgca cctggtgttg cgcctccgcg gtggtatggt gagcaagggc gaggagctgt
      421 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
      481 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
      541 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
      601 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
      661 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
      721 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
      781 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
      841 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
      901 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
      961 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga
     1021 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     1081 ggatcactct cggcatggac gagctgtaca agtaccacta cctgcccttc agcctgcccc
     1141 accggaggcc tcacttcttc tttcccaagt cccgcatcgt ccgctaagct tagatccggc
     1201 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     1261 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     1321 atccggatat cccgcaagag gcccggcagt accggcataa ccaagcctat gcctacagca
     1381 tccagggtga cggtgccgag gatgacgatg agcgcattgt tagatttcat acacggtgcc
     1441 tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt aaagctaatt cttgaagacg
     1501 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     1561 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1621 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     1681 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     1741 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     1801 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     1861 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     1921 tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     1981 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     2041 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     2101 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     2161 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     2221 gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga
     2281 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     2341 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     2401 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     2461 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     2521 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     2581 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     2641 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     2701 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     2761 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2821 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     2881 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     2941 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     3001 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     3061 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     3121 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     3181 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3241 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3301 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3361 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3421 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3481 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3541 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     3601 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     3661 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3721 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     3781 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     3841 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     3901 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     3961 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     4021 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     4081 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     4141 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     4201 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     4261 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     4321 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     4381 gcacgatcat gcgcacccgt ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc
     4441 tgctggagat ggcggacgcg atggatatgt tctgccaagg gttggtttgc gcattcacag
     4501 ttctccgcaa gaattgattg gctccaattc ttggagtggt gaatccgtta gcgaggtgcc
     4561 gccggcttcc attcaggtcg aggtggcccg gctccatgca ccgcgacgca acgcggggag
     4621 gcagacaagg tatagggcgg cgcctacaat ccatgccaac ccgttccatg tgctcgccga
     4681 ggcggcataa atcgccgtga cgatcagcgg tccaatgatc gaagttaggc tggtaagagc
     4741 cgcgagcgat ccttgaagct gtccctgatg gtcgtcatct acctgcctgg acagcatggc
     4801 ctgcaacgcg ggcatcccga tgccgccgga agcgagaaga atcataatgg ggaaggccat
     4861 ccagcctcgc gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg ccatgccggc
     4921 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     4981 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     5041 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     5101 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     5161 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     5221 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5281 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     5341 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     5401 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     5461 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     5521 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     5581 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     5641 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     5701 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     5761 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     5821 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     5881 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     5941 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     6001 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     6061 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     6121 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     6181 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     6241 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     6301 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     6361 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     6421 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     6481 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     6541 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     6601 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     6661 atgccggcca cgatgcgtcc ggcgtagagg atcg
//
