LOCUS       sequence-201748-        8490 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human UBA1 tagged with His.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8490)
  AUTHORS   Pichler A, Knipscheer P, Oberhofer E, van Dijk WJ, Korner R, Olsen 
            JV, Jentsch S, Melchior F, Sixma TK
  TITLE     SUMO modification of the ubiquitin-conjugating enzyme E2-25K.
  JOURNAL   Nat Struct Mol Biol. 2005 Mar;12(3):264-9. Epub 2005 Feb 20.
  PUBMED    15723079
REFERENCE   2  (bases 1 to 8490)
  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: "Nat Struct 
            Mol Biol."; date: "2005-03"; volume: "12(3)"; pages: "264-9. Epub 
            2005 Feb 20"
COMMENT     Sequence Label: pET3a-hUBA1
FEATURES             Location/Qualifiers
     source          1..8490
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3143..3172
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             3222..3239
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3292..3310)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3306..3353
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3390..3845
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3477..3496)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3687..3708
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3871..3975
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3976..4044,4045..4836)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4194..4213)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5007..5595
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5496..5515
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5749..5766
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5781..5920
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5846..5865)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6006..6028
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6022..6213)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7022..8104)
                     /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     8066..8085
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8105..8182)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8388..8407
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat ggccaagaac ggcagtgaag cagacataga
      121 cgagggcctt tactcccggc agctgtatgt gttgggccat gaggcaatga agcggctcca
      181 gacatccagt gtcctggtat caggcctgcg gggcctgggc gtggagatcg ctaagaacat
      241 catccttggt ggggtcaagg ctgttaccct acatgaccag ggcactgccc agtgggctga
      301 tctttcctcc cagttctacc tgcgggagga ggacatcggt aaaaaccggg ccgaggtatc
      361 acagccccgc ctcgctgagc tcaacagcta tgtgcctgtc actgcctaca ctggacccct
      421 cgttgaggac ttccttagtg gtttccaggt ggtggtgctc accaacaccc ccctggagga
      481 ccagctgcga gtgggtgagt tctgtcacaa ccgtggcatc aagctggtgg tggcagacac
      541 gcggggcctg tttgggcaac tcttctgtga ctttggagag gaaatgatcc tcacagattc
      601 caatggggag cagccactca gtgctatggt ttctatggtt accaaggaca accccggtgt
      661 ggttacctgc ctggatgagg cccgacacgg gtttgagagc ggggactttg tctccttttc
      721 agaagtacag ggcatggttg aactcaacgg aaatcagccc atggagatca aagtcctggg
      781 tccttatacc tttagcatct gtgacacctc caacttctcc gactacatcc gtggaggcat
      841 cgtcagtcag gtcaaagtac ctaagaagat tagctttaaa tccttggtgg cctcactggc
      901 agaacctgac tttgtggtga cggacttcgc caagttttct cgccctgccc agctgcacat
      961 tggcttccag gccctgcacc agttctgtgc tcagcatggc cggccacccc ggccccgcaa
     1021 tgaggaggat gcagcagaac tggtagcctt agcacaggct gtgaatgctc gagccctgcc
     1081 agcagtgcag caaaataacc tggacgagga cctcatccgg aagctggcat atgtggctac
     1141 tggggatctg gcacccataa acgccttcat tgggggcctg gctgcccagg aagtcatgaa
     1201 ggcctgctcc gggaagttca tgcccatcat gcagtggcta tactttgatg cccttgagtg
     1261 tctccctgtg gacaaagagg tcctcacaga agacaagtgc ctccagcgcc agaaccgtta
     1321 tgacgggcaa gtggctgtgt ttggctcaga cctgcaagag aagctgggca agcagaagta
     1381 tttcctggtg ggtgcggggg ccattggctg tgagctgctc aagaactttg ccatgattgg
     1441 gctgggctgc ggggagggtg gagaaatcat cgttacagac atggacacca ttgagaagtc
     1501 aaatctgaat cgacagtttc ttttccggcc ctgggatgtc acgaagttaa agtctgacac
     1561 ggctgctgca gctgtgcgcc aaatgaatcc acatatccgg gtgacaagcc accagaaccg
     1621 tgtgggtcct gacacggagc gcatctatga tgacgatttt ttccaaaacc tagatggcgt
     1681 ggccaatgcc ctggacaacg tggatgcccg catgtacatg gaccgccgct gtgtctacta
     1741 ccggaagcca ctgctggagt caggcacact gggcaccaaa ggcaatgtgc aggtggtgat
     1801 ccccttcctg acagagtcgt acagttccag ccaggaccca cctgagaagt ccatccccat
     1861 ctgtaccctg aagaacttcc ctaatgccat cgagcacacc ctgcagtggg ctcgggatga
     1921 gtttgaaggc ctcttcaagc agccagcaga aaatgtcaac cagtacctca cagaccccaa
     1981 gtttgtggag cgaacactgc ggctggcagg cactcagccc ttggaggtgc tggaggctgt
     2041 gcagcgcagc ctggtgctgc agcgaccaca gacctgggct gactgcgtga cctgggcctg
     2101 ccaccactgg cacacccagt actcgaacaa catccggcag ctgctgcaca acttccctcc
     2161 tgaccagctc acaagctcag gagcgccgtt ctggtctggg cccaaacgct gtccacaccc
     2221 gctcaccttt gatgtcaaca atcccctgca tctggactat gtgatggctg ctgccaacct
     2281 gtttgcccag acctacgggc tgacaggctc tcaggaccga gctgctgtgg ccacattcct
     2341 gcagtctgtg caggtccccg aattcacccc caagtctggc gtcaagatcc atgtttctga
     2401 ccaggagctg cagagcgcca atgcctctgt tgatgacagt cgtctagagg agctcaaagc
     2461 cactctgccc agcccagaca agctccctgg attcaagatg taccccattg actttgagaa
     2521 ggatgatgac agcaactttc atatggattt catcgtggct gcatccaacc tccgggcaga
     2581 aaactatgac attccttctg cagaccggca caagagcaag ctgattgcag ggaagatcat
     2641 cccagccatt gccacgacca cagcagccgt ggttggcctt gtgtgtctgg aactgtacaa
     2701 ggttgtgcag gggcaccgac agcttgactc ctacaagaat ggtttcctca acttggccct
     2761 gcctttcttt ggtttctctg aaccccttgc cgcaccacgt caccagtact ataaccaaga
     2821 gtggacattg tgggatcgct ttgaggtaca agggctgcag cctaatggtg aggagatgac
     2881 cctcaaacag ttcctcgact attttaagac agagcacaaa ttagagatca ccatgctgtc
     2941 ccagggcgtg tccatgctct attccttctt catgccagct gccaagctca aggaacggtt
     3001 ggatcagccg atgacagaga ttgtgagccg tgtgtcgaag cgaaagctgg gccgccacgt
     3061 gcgggcgctg gtgcttgagc tgtgctgtaa cgacgagagc ggcgaggatg tcgaggttcc
     3121 ctatgtccga tacaccatcc gccatcacca tcaccatcac catcaccatc actaaggatc
     3181 cgaattcgag ctccgtcgac aagcttgcgg ccgcactcga gcaccaccac caccaccact
     3241 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     3301 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     3361 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     3421 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3481 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3541 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3601 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3661 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3721 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3781 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     3841 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     3901 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     3961 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     4021 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     4081 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     4141 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     4201 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     4261 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     4321 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     4381 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     4441 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     4501 cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa
     4561 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     4621 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     4681 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     4741 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     4801 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     4861 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     4921 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     4981 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5041 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     5101 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     5161 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     5221 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     5281 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     5341 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     5401 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     5461 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     5521 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     5581 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     5641 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     5701 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     5761 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     5821 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     5881 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     5941 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     6001 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     6061 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     6121 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     6181 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     6241 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     6301 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     6361 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     6421 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     6481 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     6541 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     6601 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     6661 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     6721 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     6781 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     6841 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     6901 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     6961 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     7021 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     7081 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     7141 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     7201 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     7261 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     7321 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     7381 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     7441 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     7501 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     7561 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     7621 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     7681 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     7741 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     7801 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     7861 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     7921 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     7981 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     8041 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     8101 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     8161 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     8221 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     8281 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     8341 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     8401 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     8461 tagaggatcg agatctcgat cccgcgaaat
//
