LOCUS       sequence-171215-        5863 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Ubiquitin G76C for cross linking studies.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5863)
  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 5863)
  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 G76C
FEATURES             Location/Qualifiers
     source          1..5863
                     /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             2680..2907
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MAQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLI
                     FAGKQLEDGRTLSDYNIQKESTLHLVLRLRG"
     terminator      2949..3035
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3127..3154
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3182..3272
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3273..3341,3342..4133)
                     /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(3491..3510)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4221..4809
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4710..4729
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4963..4980
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5163..5185
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5179..5370)
                     /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 tgctgctgac
     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 tttcagtcca tggctcagat cttcgtcaag
     2701 acgttaaccg gtaaaaccat aactctggaa gttgaaccat ccgataccat cgaaaacgtt
     2761 aaggctaaaa ttcaagacaa ggaaggaatt ccacctgatc aacaaagatt gatctttgcc
     2821 ggtaagcagc tcgaggacgg tagaacgctg tctgattaca acattcagaa ggagtcgacc
     2881 ttacatcttg tcttaagact aagaggttgc taaggatccg aattccctgc aggtaattaa
     2941 ataagcttca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     3001 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg
     3061 cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa
     3121 ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact ctttcggtcc
     3181 gttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     3241 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     3301 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     3361 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     3421 acagcggtaa gatccttgag agttttcgcc ccgaagaacg tttcccaatg atgagcactt
     3481 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgcctggcaa gagcaactcg
     3541 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     3601 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     3661 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     3721 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     3781 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     3841 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     3901 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     3961 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     4021 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     4081 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     4141 accaagttta ctcatatata ctttagattg atttccttag gactgagcgt caaccccgta
     4201 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     4261 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4321 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     4381 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4441 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4501 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4561 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4621 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4681 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4741 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4801 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4861 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4921 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4981 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     5041 cgcatataag gtgcactgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     5101 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     5161 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     5221 gcggtaaagc tcatcagcgt ggtcgtgcag cgattcacag atgtctgcct gttcatccgc
     5281 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     5341 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     5401 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     5461 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     5521 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     5581 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     5641 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     5701 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     5761 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     5821 gatcatgcgc acccgtggcc aggacccaac gctgcccgaa att
//
