LOCUS       sequence-472389-        5765 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express a fragment of the double mutant of the human UBC9 (generated
            from pET28-UBC9 through mutagenesis; point mutations: K14R and 
            A129K).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5765)
  TITLE     Production and purification of SUMO-UBC9 and SUMO-RANGAP1CTD
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5765)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pET28a-UBC9-K14R-A129K
FEATURES             Location/Qualifiers
     source          1..5765
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(638..655)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(665..682)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             695..717
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    732..756
                     /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)."
     primer_bind     complement(756..775)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(757..775)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(859..878)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1084..1161
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1162..2244
                     /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     complement(1181..1200)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3053..3244
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3238..3260)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3346..3485
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3401..3420
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3500..3517)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3682..4270)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3762..3781)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4381..4400)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4392..5207
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4392..5207
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4465..4484)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5299..5754)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5436..5457)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5648..5667
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagttatgag ggcgcaaact
      181 tcttggcttg tgctcggacc cttttctcgt actccactct gttttggcag taaatcgtgt
      241 aggcctctgc ttgctttggg tcttggatat ttggttcatt tagaagttcc tgtattccta
      301 ataggatctg tttgattgtg atggctggcc tccagtcctt gtcctcctct aagatggaca
      361 ggcacactgt ccccgaaggg tacacattcg ggtgaaataa tggtggttcg aatttacatt
      421 ttggtggcga agatggataa tcatctttga aaagcatccg tagtttaaac aagcctcctt
      481 cccacggagt ccctttcttt cctggaatgg cgcactccca gttcatgagg ttcatcgtgc
      541 catcgggatt ttttgttggg acagccacga aaccaaatgg gtggtctttc ctccatgctc
      601 tcctctcctg ggcgagtctg ctgagggcga tccccgagct gccgcgcggc accaggccgc
      661 tgctgtgatg atgatgatga tggcccatgg tatatctcct tcttaaagtt aaacaaaatt
      721 atttctagag gggaattgtt atccgctcac aattccccta tagtgagtcg tattaatttc
      781 gcgggatcga gatctcgatc ctctacgccg gacgcatcgt ggccggcatc accggcgcca
      841 caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc
      901 acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg
      961 gactgttggg cgccatctcc ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc
     1021 tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag cgtcgagatc
     1081 ccggacacca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag
     1141 agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc
     1201 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa
     1261 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca
     1321 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg
     1381 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc
     1441 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat
     1501 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc
     1561 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag
     1621 acacccatca acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat
     1681 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg
     1741 gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata
     1801 gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg
     1861 aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca
     1921 atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac
     1981 gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt
     2041 cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg
     2101 aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat
     2161 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     2221 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc tcactcatta
     2281 ggcaccggga tctcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg
     2341 ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt
     2401 aggacaggtg ccggcagcgc tctgggtcat tttcggcgag gaccgctttc gctggagcgc
     2461 gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt
     2521 cgtcactggt cccgccacca aacgtttcgg cgagaagcag gccattatcg ccggcatggc
     2581 ggccccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg
     2641 ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg
     2701 ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta
     2761 aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg
     2821 atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc
     2881 ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg
     2941 ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt
     3001 catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa
     3061 acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat taacgcttct
     3121 ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga
     3181 ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct
     3241 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     3301 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca
     3361 gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta
     3421 ctgagagtgc accattgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
     3481 tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     3541 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     3601 caggaaagaa catgtgagca aaaggccagc aaaaggccag caaaaggcca ggaaccgtaa
     3661 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     3721 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     3781 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     3841 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     3901 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     3961 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4021 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4081 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     4141 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     4201 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     4261 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     4321 ctcacgttaa gggattttgg tcatgaacaa taaaactgtc tgcttacata aacagtaata
     4381 caaggggtgt tatgagccat attcaacggg aaacgtcttg ctctaggccg cgattaaatt
     4441 ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag
     4501 gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg
     4561 gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg
     4621 aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac
     4681 tcaccactgc gatccccggg aaaacagcat tccaggtatt agaagaatat cctgattcag
     4741 gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt
     4801 gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga
     4861 ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac
     4921 aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg
     4981 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg
     5041 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg
     5101 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg
     5161 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaagaa ttaattcatg
     5221 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5281 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     5341 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     5401 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     5461 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     5521 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     5581 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     5641 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     5701 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
     5761 cgcca
//
