LOCUS       sequence-181112-        8609 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8609)
  AUTHORS   Berndsen CE, Wolberger C
  TITLE     A spectrophotometric assay for conjugation of ubiquitin and 
            ubiquitin-like proteins.
  JOURNAL   Anal Biochem. 2011 Nov 1;418(1):102-10. Epub 2011 Jul 2.
  PUBMED    21771579
REFERENCE   2  (bases 1 to 8609)
  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: "Anal 
            Biochem."; date: "2011-11-1"; volume: "418(1)"; pages: "102-10. Epub
            2011 Jul 2"
COMMENT     Sequence Label: Ube1/PET21d
FEATURES             Location/Qualifiers
     source          1..8609
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             302..319
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3549..3566
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3619..3637)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3633..3680
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3717..4172
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3804..3823)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4014..4035
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4198..4302
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4303..4371,4372..5163)
                     /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(4521..4540)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5334..5922
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5823..5842
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6076..6093
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6108..6247
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6173..6192)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6333..6355
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6349..6540)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7349..8431)
                     /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     8393..8412
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8432..8509)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatggg
      301 tcatcaccat catcatcacg gaggtatgtc cagctcgccg ctgtccaaga aacgtcgcgt
      361 gtccgggcct gatccaaagc cgggttctaa ctgctcccct gcccagtccg tgttgtccga
      421 agtgccctcg gtgccaacca acggaatggc caagaacggc agtgaagcag acatagacga
      481 gggcctttac tcccggcagc tgtatgtgtt gggccatgag gcaatgaagc ggctccagac
      541 atccagtgtc ctggtatcag gcctgcgggg cctgggcgtg gagatcgcta agaacatcat
      601 ccttggtggg gtcaaggctg ttaccctaca tgaccagggc actgcccagt gggctgatct
      661 ttcctcccag ttctacctgc gggaggagga catcggtaaa aaccgggccg aggtatcaca
      721 gccccgcctc gctgagctca acagctatgt gcctgtcact gcctacactg gacccctcgt
      781 tgaggacttc cttagtggtt tccaggtggt ggtgctcacc aacacccccc tggaggacca
      841 gctgcgagtg ggtgagttct gtcacaaccg tggcatcaag ctggtggtgg cagacacgcg
      901 gggcctgttt gggcagctct tctgtgactt tggagaggaa atgatcctca cagattccaa
      961 tggggagcag ccactcagtg ctatggtttc tatggttacc aaggacaacc ccggtgtggt
     1021 tacctgcctg gatgaggccc gacacgggtt tgagagcggg gactttgtct ccttttcaga
     1081 agtacagggc atggttgaac tcaacggaaa tcagcccatg gagatcaaag tcctgggtcc
     1141 ttataccttt agcatctgtg acacctccaa cttctccgac tacatccgtg gaggcatcgt
     1201 cagtcaggtc aaagtaccta agaagattag ctttaaatcc ttggtggcct cactggcaga
     1261 acctgacttt gtggtgacgg acttcgccaa gttttctcgc cctgcccagc tgcacattgg
     1321 cttccaggcc ctgcaccagt tctgtgctca gcatggccgg ccacctcggc cccgcaatga
     1381 ggaggatgca gcagaactgg tagccttagc acaggctgtg aatgctcgag ccctgccagc
     1441 agtgcagcaa aataacctgg acgaggacct catccggaag ctggcatatg tggctgctgg
     1501 ggatctggca cccataaacg ccttcattgg gggcctggct gcccaggaag tcatgaaggc
     1561 ctgctccggg aagttcatgc ccatcatgca gtggctatac tttgatgccc ttgagtgtct
     1621 ccctgaggac aaagaggtcc tcacagagga caagtgcctc cagcgccaga accgttatga
     1681 cgggcaagtg gctgtgtttg gctcagacct gcaagagaag ctgggcaagc agaagtattt
     1741 cctggtgggt gcgggggcca ttggctgtga gctgctcaag aactttgcca tgattgggct
     1801 gggctgcggg gagggtggag aaatcatcgt tacagacatg gacaccattg agaagtcaaa
     1861 tctgaatcga cagtttcttt tccggccctg ggatgtcacg aagttaaagt ctgacacggc
     1921 tgctgcagct gtgcgccaaa tgaatccaca tatccgggtg acaagccacc agaaccgtgt
     1981 gggtcctgac acggagcgca tctatgatga cgattttttc caaaacctag atggcgtggc
     2041 caatgccctg gacaacgtgg atgcccgcat gtacatggac cgccgctgtg tctactaccg
     2101 gaagccactg ctggagtcag gcacactggg caccaaaggc aatgtgcagg tggtgatccc
     2161 cttcctgaca gagtcgtaca gttccagcca ggacccacct gagaagtcca tccccatctg
     2221 taccctgaag aacttcccta atgccatcga gcacaccctg cagtgggctc gggatgagtt
     2281 tgaaggcctc ttcaagcagc cagcagaaaa tgtcaaccag tacctcacag accccaagtt
     2341 tgtggagcga acactgcggc tggcaggcac tcagcccttg gaggtgctgg aggctgtgca
     2401 gcgcagcctg gtgctgcagc gaccacagac ctgggctgac tgcgtgacct gggcctgcca
     2461 ccactggcac acccagtact cgaacaacat ccggcagctg ctgcacaact tccctcctga
     2521 ccagctcaca agctcaggag cgccgttctg gtctgggccc aaacgctgtc cacacccgct
     2581 cacctttgat gtcaacaatc ccctgcatct ggactatgtg atggctgctg ccaacctgtt
     2641 tgcccagacc tacgggctga caggctctca ggaccgagct gctgtggcca cattcctgca
     2701 gtctgtgcag gtccccgaat tcacccccaa gtctggcgtc aagatccatg tttctgacca
     2761 ggagctgcag agcgccaatg cctctgttga tgacagtcgt ctagaggagc tcaaagccac
     2821 tctgcccagc ccagacaagc tccctggatt caagatgtac cccattgact ttgagaagga
     2881 tgatgacagc aactttcata tggatttcat cgtggctgca tccaacctcc gggcagaaaa
     2941 ctatgacatt ccttctgcag accggcacaa gagcaagctg attgcaggga agatcatccc
     3001 agccattgcc acgaccacag cagccgtggt tggccttgtg tgtctggagc tgtacaaggt
     3061 tgtgcagggg caccgacagc ttgactccta caagaatggt ttcctcaact tggccctgcc
     3121 tttctttggt ttctctgaac cccttgccgc accacgtcac cagtactata accaagagtg
     3181 gacattgtgg gatcgctttg aggtacaagg gctgcagcct aatggtgagg agatgaccct
     3241 caaacagttc ctcgactatt ttaagacaga gcacaaatta gagatcacca tgctgtccca
     3301 gggcgtgtcc atgctctatt ccttcttcat gccagctgcc aagctcaagg aacggttgga
     3361 tcagccgatg acagagattg tgagccgtgt gtcgaagcga aagctgggcc gccacgtgcg
     3421 ggcgctggtg cttgagctgt gctgtaacga cgagagcggc gaggatgtcg aggttcccta
     3481 tgtccgatac accatccgct gaggatccga attcgagctc cgtcgacaag cttgcggccg
     3541 cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag
     3601 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     3661 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     3721 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3781 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3841 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3901 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     3961 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4021 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     4081 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4141 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     4201 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     4261 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     4321 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     4381 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     4441 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     4501 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     4561 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     4621 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     4681 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     4741 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     4801 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca
     4861 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     4921 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     4981 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     5041 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     5101 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     5161 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     5221 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     5281 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     5341 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     5401 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     5461 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     5521 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     5581 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     5641 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     5701 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     5761 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     5821 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     5881 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     5941 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     6001 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     6061 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     6121 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     6181 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     6241 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     6301 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     6361 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     6421 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     6481 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     6541 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     6601 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     6661 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     6721 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     6781 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     6841 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     6901 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     6961 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     7021 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     7081 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     7141 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     7201 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     7261 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     7321 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     7381 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     7441 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     7501 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     7561 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     7621 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     7681 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     7741 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     7801 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     7861 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     7921 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     7981 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     8041 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     8101 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     8161 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     8221 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     8281 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     8341 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     8401 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     8461 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     8521 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     8581 agcaccgccg ccgcaaggaa tggtgcatg
//
