LOCUS       sequence-210944-        6984 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression in E. coli of full-length SPRTN protein carrying E112A 
            mutation, a catalytically-dead mutant, with His and ZB tags at 
            N-terminus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6984)
  AUTHORS   Vaz B, Popovic M, Newman JA, Fielden J, Aitkenhead H, Halder S, 
            Singh AN, Vendrell I, Fischer R, Torrecilla I, Drobnitzky N, Freire 
            R, Amor DJ, Lockhart PJ, Kessler BM, McKenna GW, Gileadi O, Ramadan 
            K
  TITLE     Metalloprotease SPRTN/DVC1 Orchestrates Replication-Coupled 
            DNA-Protein Crosslink Repair.
  JOURNAL   Mol Cell. 2016 Nov 17;64(4):704-719. doi: 
            10.1016/j.molcel.2016.09.032. Epub 2016 Oct 27.
  PUBMED    27871366
REFERENCE   2  (bases 1 to 6984)
  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"; doi: 
            "10.1016/j.molcel.2016.09.032"; journalName: "Mol Cell"; date: 
            "2016-11-17- 17"; volume: "64"; issue: "4"; pages: "704-719"
COMMENT     Sequence Label: pNIC-ZB-SPRTN-E112A-FL
FEATURES             Location/Qualifiers
     source          1..6984
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     61..80
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        61..79
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    80..104
                     /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             119..141
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             152..169
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             179..349
                     /codon_start=1
                     /product="Z domain of staphylococcal protein A, engineered 
                     to contain multiple positive charges (Hedhammar and Hober, 
                     2007)"
                     /label=Z-basic tag
                     /note="useful for purifying tagged proteins by 
                     cation-exchange chromatography"
                     /translation="VDNKFNKERRRARREIRHLPNLNREQRRAFIRSLRDDPSQSANLL
                     AEAKKLNDAQPK"
     CDS             356..376
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1909..1926
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1979..1997)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1993..2040
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2077..2532
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2164..2183)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2374..2395
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2624..3439)
                     /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             complement(2624..3439)
                     /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     3347..3366
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3431..3450
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3561..4149
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4050..4069
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4303..4320
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4335..4474
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4400..4419)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4560..4582
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4576..4767)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5576..6658)
                     /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     6620..6639
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6659..6736)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6942..6961
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
       61 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
      121 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt
      181 ggataacaag ttcaacaagg agcgtcgaag agctcgccgt gaaattcgcc atctgccgaa
      241 cctgaaccgc gaacagcgtc gcgcatttat tcgcagcctg cgcgatgatc cgagccagag
      301 cgcgaacctg ctggcggaag cgaagaagct gaacgatgcg cagccgaagg gtaccgagaa
      361 cctgtacttc caatccatgg atgatgactt gatgttggca ctgcggcttc aggaggagtg
      421 gaacttgcag gaggcggagc gcgatcatgc ccaggagtcc ctgtcgctag tggacgcgtc
      481 gtgggagttg gtggacccca caccggactt gcaggcactg tttgttcagt ttaacgacca
      541 attcttctgg ggccagctgg aggccgtcga ggtgaagtgg agcgtgcgaa tgaccctgtg
      601 tgctgggata tgcagctatg aagggaaggg tggaatgtgt tccatccgtc tcagcgaacc
      661 ccttttgaag ttgaggccaa gaaaggatct tgtagagacc ctcctgcatg caatgataca
      721 tgcctattta tttgtcacta ataacgacaa agaccgagaa gggcatggtc cagaattttg
      781 taaacatatg catcgcatca acagcctgac tggagccaat ataacggtat accatacttt
      841 tcacgatgag gtggatgagt atcggcgaca ctggtggcgc tgcaatgggc cgtgccagca
      901 caggccaccg tattacggct atgtcaaacg agctactaac agggaaccct ctgctcatga
      961 ctattggtgg gctgagcacc agaaaacctg tggaggcact tacataaaaa tcaaggaacc
     1021 agagaattac tcaaaaaaag gcaaaggaaa ggcaaaacta ggaaaggaac cagtattggc
     1081 cgcagagaat aaagataaac ccaacagagg tgaggcccag ctagtaatcc cttttagtgg
     1141 gaaaggatat gttctaggag aaacaagcaa tttaccttca cctgggaaac tgatcacttc
     1201 acatgccatt aataaaaccc aagatctttt aaatcaaaac cattcagcaa atgctgtaag
     1261 acttaattct aaaatcaagg tgaaatttga acagaatggt tcaagtaaaa attctcatct
     1321 ggtctcccct gctgttagta acagtcacca aaatgttcta agcaactact ttcctagagt
     1381 atcatttgcc aaccaaaagg ctttcagagg tgtgaatgga tctccaagga taagtgtaac
     1441 agttggcaac atccctaaaa actcagtctc ttctagttct cagagaaggg tttcatcttc
     1501 taagatatcc ctaagaaatt cttcaaaagt aacggaatca gcatctgtga tgccatccca
     1561 ggatgtgagt gggtctgaag atacattccc aaataaacga cctaggctag aagataagac
     1621 tgtttttgac aattttttta tcaagaaaga gcaaataaaa agcagtggta atgatccaaa
     1681 gtatagtaca accacagctc agaattccag cagttcatcc agtcagagca aaatggttaa
     1741 ttgcccagtt tgtcagaatg aagttctgga gtctcagatt aatgagcact tggactggtg
     1801 ccttgaaggt gacagcatca aagtcaaaag cgaagaaagt ctttgacagt aaaggtggat
     1861 acggatccga attcgagctc cgtcgacaag cttgcggccg cactcgagca ccaccaccac
     1921 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     1981 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     2041 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     2101 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     2161 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     2221 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     2281 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     2341 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     2401 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     2461 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     2521 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     2581 tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga
     2641 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     2701 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     2761 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     2821 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     2881 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     2941 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     3001 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     3061 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     3121 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     3181 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     3241 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     3301 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     3361 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt
     3421 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
     3481 ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3541 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3601 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3661 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     3721 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3781 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3841 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3901 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3961 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4021 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4081 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4141 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4201 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4261 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4321 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4381 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     4441 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     4501 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4561 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     4621 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     4681 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     4741 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     4801 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     4861 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     4921 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     4981 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     5041 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     5101 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     5161 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     5221 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     5281 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     5341 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     5401 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     5461 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     5521 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     5581 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5641 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     5701 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     5761 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     5821 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
     5881 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     5941 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     6001 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     6061 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     6121 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     6181 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     6241 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     6301 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     6361 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     6421 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     6481 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     6541 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     6601 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     6661 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     6721 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     6781 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     6841 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     6901 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     6961 gcgccagcaa ccgcacctgt ggcg
//
