LOCUS       sequence-210941-        6468 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression in mammalian cells of of full-length SPRTN protein 
            carrying Y117C mutation, a catalytically-impaired mutant, and 
            Flag-tag at N-terminus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6468)
  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 6468)
  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: pCDNA3-Flag-SPRTN-Y117C
FEATURES             Location/Qualifiers
     source          1..6468
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        74..453
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        454..657
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     608..628
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     702..721
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        702..720
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             777..800
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2315..2332)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2321..2545
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2591..3019
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2678..2697)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2888..2909
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3028..3048)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3033..3362
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3213..3348
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3275..3294
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3410..3457
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3476..3850
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    3980..4101
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4017..4036)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4071..4090
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4150..4166)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4150..4166)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4163..4185)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4174..4190
                     /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)."
     promoter        complement(join(4198..4204,4205..4222,4223..4228))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4243..4264
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4381..4398)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4552..5140)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4632..4651)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5311..6102,6103..6171))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5934..5953
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6172..6276)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6351..6370)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
       61 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
      121 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      181 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      241 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      301 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      361 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      421 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      481 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      541 gggagtttgt tttggcaccg aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      601 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc
      661 tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg
      721 gagacccaag ctggctagcg tttaaactta agcttggtac cgagctcgga tccatggatt
      781 ataaggatga cgatgataaa atggatgatg acttgatgtt ggcactgcgg cttcaggagg
      841 agtggaactt gcaggaggcg gagcgcgatc atgcccagga gtccctgtcg ctagtggacg
      901 cgtcgtggga gttggtggac cccacaccgg acttgcaggc actgtttgtt cagtttaacg
      961 accaattctt ctggggccag ctggaggccg tcgaggtgaa gtggagcgtg cgaatgaccc
     1021 tgtgtgctgg gatatgcagc tatgaaggga agggtggaat gtgttccatc cgtctcagcg
     1081 aacccctttt gaagttgagg ccaagaaagg atcttgtaga gaccctcctg catgaaatga
     1141 tacatgcctg tttatttgtc actaataacg acaaagaccg agaagggcat ggtccagaat
     1201 tttgtaaaca tatgcatcgc atcaacagcc tgactggagc caatataacg gtataccata
     1261 cttttcacga tgaggtggat gagtatcggc gacactggtg gcgctgcaat gggccgtgcc
     1321 agcacaggcc accgtattac ggctatgtca aacgagctac taacagggaa ccctctgctc
     1381 atgactattg gtgggctgag caccagaaaa cctgtggagg cacttacata aaaatcaagg
     1441 aaccagagaa ttactcaaaa aaaggcaaag gaaaggcaaa actaggaaag gaaccagtat
     1501 tggccgcaga gaataaagat aaacccaaca gaggtgaggc ccagctagta atccctttta
     1561 gtgggaaagg atatgttcta ggagaaacaa gcaatttacc ttcacctggg aaactgatca
     1621 cttcacatgc cattaataaa acccaagatc ttttaaatca aaaccattca gcaaatgctg
     1681 taagacttaa ttctaaaatc aaggtgaaat ttgaacagaa tggttcaagt aaaaattctc
     1741 atctggtctc ccctgctgtt agtaacagtc accaaaatgt tctaagcaac tactttccta
     1801 gagtatcatt tgccaaccaa aaggctttca gaggtgtgaa tggatctcca aggataagtg
     1861 taacagttgg caacatccct aaaaactcag tctcttctag ttctcagaga agggtttcat
     1921 cttctaagat atccctaaga aattcttcaa aagtaacgga atcagcatct gtgatgccat
     1981 cccaggatgt gagtgggtct gaagatacat tcccaaataa acgacctagg ctagaagata
     2041 agactgtttt tgacaatttt tttatcaaga aagagcaaat aaaaagcagt ggtaatgatc
     2101 caaagtatag tacaaccaca gctcagaatt ccagcagttc atccagtcag agcaaaatgg
     2161 ttaattgccc agtttgtcag aatgaagttc tggagtctca gattaatgag cacttggact
     2221 ggtgccttga aggtgacagc atcaaagtca aaagcgaaga aagtctttga gcggccgctc
     2281 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     2341 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     2401 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     2461 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     2521 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     2581 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     2641 agcgtgaccg ctacacttgc cagcgcccta gcgccagctc ctttcgcttt cttcccttcc
     2701 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     2761 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     2821 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     2881 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     2941 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     3001 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3061 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     3121 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3181 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3241 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3301 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3361 aaaagctccc gggagcttgt atatccattt tcggatctga tcagcacgtg ttgacaatta
     3421 atcatcggca tagtatatcg gcatagtata atacgacaag gtgaggaact aaaccatggc
     3481 caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt
     3541 ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg ccggtgtggt
     3601 ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc cggacaacac
     3661 cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt cggaggtcgt
     3721 gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg agcagccgtg
     3781 ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg tggccgagga
     3841 gcaggactga cacgtgctac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     3901 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     3961 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     4021 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4081 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     4141 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4201 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4261 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4321 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4381 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4441 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4501 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4561 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4621 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4681 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4741 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4801 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4861 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4921 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4981 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5041 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5101 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5161 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5221 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5281 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5341 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5401 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     5461 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     5521 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     5581 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5641 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5701 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     5761 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5821 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5881 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5941 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6001 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6061 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6121 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6181 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6241 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     6301 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
     6361 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
     6421 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgac
//
