LOCUS       sequence-470411-        6667 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a His6-Ubiquitin-Clu(228-240)-eGFP fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6667)
  TITLE     Hartl lab plasmids 2024
REFERENCE   2  (bases 1 to 6667)
  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-Clu-alpha-tail-GFP
FEATURES             Location/Qualifiers
     source          1..6667
                     /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(441..443,444..446,447..1160)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(441..443,444..446,447..1157)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(486..507)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(747..766)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1094..1115
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1215..1233)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1229..1276
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1479..1497)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1565..1669
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1670..1738,1739..2530)
                     /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(1888..1907)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2701..3289
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3190..3209
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3443..3460
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3475..3614
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3540..3559)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3700..3722
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3716..3907)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5219..6301)
                     /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     6263..6282
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6302..6379)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6585..6604
                     /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 gtggtagctt gatgcccttc tctccgtacg
      421 agcccctgaa cttcggcggc atggtgagca agggcgagga gctgttcacc ggggtggtgc
      481 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
      541 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
      601 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
      661 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
      721 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
      781 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
      841 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
      901 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
      961 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     1021 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     1081 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     1141 tggacgagct gtacaagtaa gcttagatcc ggctgctaac aaagcccgaa aggaagctga
     1201 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     1261 cttgaggggt tttttgctga aaggaggaac tatatccgga tatcccgcaa gaggcccggc
     1321 agtaccggca taaccaagcc tatgcctaca gcatccaggg tgacggtgcc gaggatgacg
     1381 atgagcgcat tgttagattt catacacggt gcctgactgc gttagcaatt taactgtgat
     1441 aaactaccgc attaaagcta attcttgaag acgaaagggc ctcgtgatac gcctattttt
     1501 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     1561 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1621 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     1681 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     1741 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     1801 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     1861 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc
     1921 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     1981 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     2041 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     2101 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     2161 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat
     2221 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     2281 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     2341 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     2401 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     2461 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     2521 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     2581 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     2641 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2701 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2761 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2821 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     2881 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2941 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3001 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3061 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3121 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3181 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3241 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3301 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3361 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     3421 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     3481 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     3541 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     3601 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     3661 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     3721 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     3781 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     3841 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     3901 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     3961 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     4021 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     4081 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     4141 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     4201 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     4261 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     4321 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg
     4381 acccaacgct gcccgagatg cgccgcgtgc ggctgctgga gatggcggac gcgatggata
     4441 tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga ttggctccaa
     4501 ttcttggagt ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc
     4561 ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgcctac
     4621 aatccatgcc aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag
     4681 cggtccaatg atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg
     4741 atggtcgtca tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc
     4801 ggaagcgaga agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa
     4861 gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     4921 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     4981 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     5041 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     5101 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     5161 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     5221 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5281 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     5341 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     5401 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     5461 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     5521 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     5581 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     5641 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     5701 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     5761 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     5821 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     5881 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     5941 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     6001 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     6061 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     6121 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     6181 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     6241 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     6301 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     6361 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     6421 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     6481 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     6541 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     6601 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     6661 aggatcg
//
