LOCUS       sequence-213473-        8040 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of UBA2_ThiF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8040)
  AUTHORS   Teyra J, Kelil A, Jain S, Helmy M, Jajodia R, Hooda Y, Gu J, D'Cruz 
            AA, Nicholson SE, Min J, Sudol M, Kim PM, Bader GD, Sidhu SS
  TITLE     Large-scale survey and database of high affinity ligands for peptide
            recognition modules.
  JOURNAL   Mol Syst Biol. 2020 Dec;16(12):e9310. doi: 10.15252/msb.20199310.
  PUBMED    33438817
REFERENCE   2  (bases 1 to 8040)
  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: "Mol Syst 
            Biol."; date: "2020-12"; volume: "16(12):e9310. doi"; pages: " 
            10.15252/msb.20199310"
COMMENT     Sequence Label: pHH0103_UBA2_ThiF
FEATURES             Location/Qualifiers
     source          1..8040
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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     42..64
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             138..155
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             171..824
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     782..804
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             831..854
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             867..884
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2235..2282
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3875..4330)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4012..4033)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4224..4243
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4372..4394)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4480..4620
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4535..4554
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4635..4652)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4806..5393)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5564..6355,6356..6424))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6187..6206
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6425..6529)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6597..6615
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6638..6715
                     /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             6716..7798
                     /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(6735..6754)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag ccagtccgtt taggtgtttt cacgagcact tcaccaacaa ggaccataga
      121 ttatgaaaat cgaagaacac catcaccatc accattccag cggtaagctt atgtccccta
      181 tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc
      241 ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca
      301 aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta
      361 aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg
      421 gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat
      481 acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta
      541 gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa
      601 atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat
      661 acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg
      721 aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc
      781 agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat ctggaagttc
      841 tgttccaggg gcccctgtcc agcggtctgg ttccgcgtgg ttccggtacc gcggcccagc
      901 cggccatggc tctgtctcgc ggtctgccgc gcgaactggc cgaagccgta gctggcggcc
      961 gtgtactggt ggttggtgcg ggcggtatcg gttgcgaact gctgaagaac ctggtactga
     1021 ctggcttctc tcacattgat ctgatcgatc tggatactat tgatgtttcc aatctgaatc
     1081 gccaatttct gtttcaaaag aaacatgttg gccgttccaa agcccaggtg gcaaaagaaa
     1141 gcgtgctgca gttttacccg aaggcgaaca tcgtagctta ccacgattcc attatgaacc
     1201 cggactacaa cgtcgaattt ttccgccagt tcattctggt aatgaacgcg ctggacaacc
     1261 gtgcagctcg taaccatgtt aaccgtatgt gtctggcggc tgatgtaccg ctgatcgaat
     1321 ctggtaccgc aggttacctg ggccaagtga cgaccatcaa gaaaggtgtt accgaatgct
     1381 atgagtgtca cccgaaacct acccagcgta ccttcccggg ttgcaccatt cgtaacaccc
     1441 cgtccgagcc gattcactgc atcgtctggg ctaaatacct gttcaaccag ctgttcggtg
     1501 aagaagacgc cgaccaggaa gtgagcccgg accgcgcgga tcctgaagcg gcgtgggaac
     1561 cgacggaagc tgaagctcgc gctcgtgcgt ccaacgaaga cggtgatatc aaacgcatca
     1621 gcaccaaaga gtgggcgaaa tctactggct acgatccagt taaactgttt acgaaactgt
     1681 tcaaagacga catccgttat ctgctgacga tggacaaact gtggcgtaaa cgtaaaccgc
     1741 cggttccact ggattgggca gaagtgcaga gccagggcga agaaactaac gcatccgacc
     1801 agcagaatga gccacagctg ggcctgaaag atcagcaggt cctggatgtg aaatcttatg
     1861 cacgtctgtt ctctaaatcc atcgagactc tgcgtgttca cctggccgaa aagggtgacg
     1921 gcgcggagct gatctgggat aaagatgacc caagcgcaat ggacttcgtg acctccgcgg
     1981 cgaacctgcg tatgcacatt ttcagcatga acatgaaatc tcgtttcgac atcaaaagca
     2041 tggctggcaa catcatcccg gcaatcgcga ccactaacgc cgttatcgct ggtctgattg
     2101 ttctggaagg tctgaagatc ctgtctggca aaatcgacca gtgtcgcacc attttcctga
     2161 ataaacagcc taacccgcgt aagaaactgc tggttccgtg cgcactggac ccgccgaacg
     2221 cggccgcata ataaaccgat acaattaaag gctccttttg gagccttttt tttggagatt
     2281 ttcaacgtga aaaaattatt attcgcaatt cctttagttg ttcctttcta ttctcactcc
     2341 gctgaaactg ttgaaagttg tttagcaaaa ccccatacag aaaattcatt tactaacgtc
     2401 tggaaagacg acaaaacttt agatcgttac gctaactatg agggctgtct gtggaatgct
     2461 acaggcgttg tagtttgtac tggtgacgaa actcagtgtc tagctagagt ggcggtggct
     2521 ctggttccgg tgattttgat tatgaaaaga tggcaaacgc taataagggg gctatgaccg
     2581 aaaatgccga tgaaaacgcg ctacagtctg acgctaaagg caaacttgat tctgtcgcta
     2641 ctgattacgg tgctgctatc gatggtttca ttggtgacgt ttccggcctt gctaatggta
     2701 atggtgctac tggtgatttt gctggctcta attcccaaat ggctcaagtc ggtgacggtg
     2761 ataattcacc tttaatgaat aatttccgtc aatatttacc ttccctccct caatcggttg
     2821 aatgtcgccc ttttgtcttt ggcgctggta aaccatatga attttctatt gattgtgaca
     2881 aaataaactt attccgtggt gtctttgcgt ttcttttata tgttgccacc tttatgtatg
     2941 tattttctac gtttgctaac atactgcgta ataaggagtc ttaatcatgc cagttctttt
     3001 ggctagcgcc gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg
     3061 ggccacctcg acctgaatgg aagccggcgg cacctcgcta acggattcac cactccaaga
     3121 attggagcca atcaattctt gcggagaact gtgaatgcgc aaaccaaccc ttggcagaac
     3181 atatccatcg cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg
     3241 tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg
     3301 ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc
     3361 tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg
     3421 taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca
     3481 ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga
     3541 ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa
     3601 cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt
     3661 ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc
     3721 aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt
     3781 ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac
     3841 gaccacgctg atgagcttta ccgcaggatc cgcgaaattg taagcgttaa tattttgtta
     3901 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     3961 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     4021 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     4081 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     4141 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     4201 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     4261 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     4321 cagggcgcgt cgcggatcct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     4381 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     4441 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg
     4501 tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga
     4561 gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     4621 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4681 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4741 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4801 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4861 aggtggcgaa acccgacagg actataaaga taccagggtt tccccctgga agctccctcg
     4921 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4981 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5041 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5101 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5161 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5221 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     5281 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5341 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5401 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5461 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5521 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5581 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5641 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5701 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5761 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5821 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgctg
     5881 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5941 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6001 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6061 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6121 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6181 cacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6241 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6301 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6361 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6421 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6481 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6541 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     6601 ggcgtatcac gaggcccttt cgtcttcaag aattcccgac accatcgaat ggtgcaaaac
     6661 ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa
     6721 accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg
     6781 cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat
     6841 ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt
     6901 gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc
     6961 gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag
     7021 cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct
     7081 gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa
     7141 tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc
     7201 ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat
     7261 cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca
     7321 taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc
     7381 catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat
     7441 gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct
     7501 gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta
     7561 tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga
     7621 ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc
     7681 actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt
     7741 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     7801 gcaacgcaat taatgtaagt tagctcactc attaggcaca attctcatgt ttgacagctt
     7861 atcatcgact gcacggtgca ccaatgcttc tggcgtcagg cagccatcgg aagctgtggt
     7921 atggctgtgc aggtcgtaaa tcactgcata attcgtgtcg ctcaaggcgc actcccgttc
     7981 tggataatgt tttttgcgcc gacatcataa cggttctggc aaatattctg aaatgagctg
//
