LOCUS       sequence-171441-        5860 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Ubiquitin K63C for cross linking studies.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5860)
  AUTHORS   Liu J, Chen Q, Rozovsky S
  TITLE     Utilizing Selenocysteine for Expressed Protein Ligation and 
            Bioconjugations.
  JOURNAL   J Am Chem Soc. 2017 Feb 10. doi: 10.1021/jacs.6b10991.
  PUBMED    28186733
REFERENCE   2  (bases 1 to 5860)
  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: "J Am Chem 
            Soc. 2017 Feb 10. doi: 10.1021/jacs.6b10991."
COMMENT     Sequence Label: Ubiquitin K63C
FEATURES             Location/Qualifiers
     source          1..5860
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        3..80
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             81..1163
                     /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(100..119)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1406..1411,1412..1427,1428..1434)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1442..1458
                     /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     1447..1469
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1537..1554
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     2620..2643
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2659..2679
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2683..2907
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="QIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFA
                     GKQLEDGRTLSDYNIQCESTLHLVLRLRGG"
     terminator      2946..3032
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3124..3151
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3179..3269
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3270..3338,3339..4130)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAWQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3488..3507)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4218..4806
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4707..4726
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4960..4977
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5160..5182
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5176..5367)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
       61 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
      121 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
      181 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
      241 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
      301 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
      361 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
      421 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
      481 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
      541 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
      601 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
      661 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
      721 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
      781 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
      841 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatttcggta gtgggatacg
      901 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
      961 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     1021 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     1081 cacaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     1141 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag
     1201 gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg
     1261 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg
     1321 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
     1381 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga
     1441 attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga
     1501 gcaattgacc aacaaggacc atagattatg aaaatccatc accatcacca tcacgaagaa
     1561 ggtaaactgg taatctggat taacggcgat aaaggctata acggtctcgc tgaagtcggt
     1621 aagaaattcg agaaagatac cggaattaaa gtcaccgttg agcatccgga taaactggaa
     1681 gagaaattcc cacaggttgc ggcaactggc gatggccctg acattatctt ctgggcacac
     1741 gaccgctttg gtggctacgc tcaatctggc ctgttggctg aaatcacccc ggacaaagcg
     1801 ttccaggaca agctgtatcc gtttacctgg gatgccgtac gttacaacgg caagctgatt
     1861 gcttacccga tcgctgttga agcgttatcg ctgatttata acaaagatct gctgccgaac
     1921 ccgccaaaaa cctgggaaga gatcccggcg ctggataaag aactgaaagc gaaaggtaag
     1981 agcgcgctga tgttcaacct gcaagaaccg tacttcacct ggccgctgat tactgctgac
     2041 gggggttatg cgttcaagta tgaaaacggc aagtacgaca ttaaagacgt gggcgtggat
     2101 aacgctggcg cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa caaacacatg
     2161 aatgcagaca ccgattactc catcgcagaa gctgccttta ataaaggcga aacagcgatg
     2221 accatcaacg gcccgtgggc atggtccaac atcgacacca gcaaagtgaa ttatggtgta
     2281 acggtactgc cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca
     2341 ggtattaacg ccgccagtcc gaacaaagag ctggcaaaag agttcctcga aaactatctg
     2401 ctgactgatg aaggtctgga agcggttaat aaagacaaac cgctgggtgc cgtagcgctg
     2461 aagtcttacg aggaagagtt ggtgaaagat ccgcgtattg ccgccactat ggaaaacgcc
     2521 cagaaaggtg aaatcatgcc gaacatcccg cagatgtccg ctttctggta tgccgtgcgt
     2581 actgcggtga tcaacgccgc cagcggtcgt cagactgtcg atgaagccct gaaagacgcg
     2641 cagactaatt cgagctctga aaacctgtat tttcagtccg ctcagatctt cgtcaagacg
     2701 ttaaccggta aaaccataac tctggaagtt gaaccatccg ataccatcga aaacgttaag
     2761 gctaaaattc aagacaagga aggaattcca cctgatcaac aaagattgat ctttgccggt
     2821 aagcagctcg aggacggtag aacgctgtct gattacaaca ttcagtgcga gtcgacctta
     2881 catcttgtct taagactaag aggtggttaa ggatccgaat tccctgcagg taattaaata
     2941 agcttcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
     3001 tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga
     3061 agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta
     3121 agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt tcggtccgtt
     3181 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     3241 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     3301 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     3361 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     3421 gcggtaagat ccttgagagt tttcgccccg aagaacgttt cccaatgatg agcactttta
     3481 aagttctgct atgtggcgcg gtattatccc gtgttgacgc ctggcaagag caactcggtc
     3541 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     3601 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     3661 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     3721 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     3781 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     3841 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     3901 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     3961 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     4021 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     4081 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     4141 aagtttactc atatatactt tagattgatt tccttaggac tgagcgtcaa ccccgtagaa
     4201 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4261 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4321 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     4381 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4441 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4501 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4561 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4621 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4681 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4741 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4801 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4861 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4921 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4981 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     5041 atataaggtg cactgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     5101 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     5161 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     5221 gtaaagctca tcagcgtggt cgtgcagcga ttcacagatg tctgcctgtt catccgcgtc
     5281 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     5341 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     5401 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     5461 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     5521 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     5581 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     5641 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     5701 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     5761 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     5821 catgcgcacc cgtggccagg acccaacgct gcccgaaatt
//
