LOCUS       sequence-470416-        6733 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a His6-Ubiquitin-Clu(228-262)-eGFP fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6733)
  TITLE     Hartl lab plasmids 2024
REFERENCE   2  (bases 1 to 6733)
  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-H7-GFP
FEATURES             Location/Qualifiers
     source          1..6733
                     /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(507..509,510..512,513..1226)
                     /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(507..509,510..512,513..1223)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(552..573)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(813..832)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1160..1181
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1281..1299)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1295..1342
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1545..1563)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1631..1735
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1736..1804,1805..2596)
                     /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(1954..1973)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2767..3355
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3256..3275
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3509..3526
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3541..3680
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3606..3625)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3766..3788
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3782..3973)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5285..6367)
                     /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     6329..6348
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6368..6445)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6651..6670
                     /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 cttccacgcc atgttccagc ccttccttga gatgatacac gaggctcagc
      481 aggccatgga catccacttc ggcggcatgg tgagcaaggg cgaggagctg ttcaccgggg
      541 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
      601 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
      661 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
      721 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
      781 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
      841 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
      901 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
      961 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     1021 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     1081 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     1141 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     1201 tcggcatgga cgagctgtac aagtaagctt agatccggct gctaacaaag cccgaaagga
     1261 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     1321 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatatc ccgcaagagg
     1381 cccggcagta ccggcataac caagcctatg cctacagcat ccagggtgac ggtgccgagg
     1441 atgacgatga gcgcattgtt agatttcata cacggtgcct gactgcgtta gcaatttaac
     1501 tgtgataaac taccgcatta aagctaattc ttgaagacga aagggcctcg tgatacgcct
     1561 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     1621 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1681 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     1741 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     1801 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     1861 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     1921 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     1981 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     2041 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     2101 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     2161 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     2221 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
     2281 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     2341 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     2401 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     2461 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     2521 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     2581 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     2641 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     2701 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     2761 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     2821 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     2881 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     2941 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3001 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3061 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3121 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3181 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3241 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3301 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3361 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3421 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     3481 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     3541 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     3601 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     3661 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     3721 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     3781 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     3841 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     3901 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     3961 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     4021 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     4081 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     4141 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     4201 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     4261 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     4321 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     4381 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     4441 gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct gctggagatg gcggacgcga
     4501 tggatatgtt ctgccaaggg ttggtttgcg cattcacagt tctccgcaag aattgattgg
     4561 ctccaattct tggagtggtg aatccgttag cgaggtgccg ccggcttcca ttcaggtcga
     4621 ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt atagggcggc
     4681 gcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa tcgccgtgac
     4741 gatcagcggt ccaatgatcg aagttaggct ggtaagagcc gcgagcgatc cttgaagctg
     4801 tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg gcatcccgat
     4861 gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg tcgcgaacgc
     4921 cagcaagacg tagcccagcg cgtcggccgc catgccggcg ataatggcct gcttctcgcc
     4981 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     5041 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     5101 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     5161 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     5221 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     5281 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5341 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     5401 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     5461 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     5521 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     5581 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     5641 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     5701 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     5761 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     5821 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     5881 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     5941 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     6001 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     6061 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     6121 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     6181 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     6241 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     6301 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     6361 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     6421 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     6481 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     6541 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     6601 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     6661 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     6721 gcgtagagga tcg
//
