LOCUS       sequence-474134-        5771 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of recombinant BMPS K319E.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5771)
  AUTHORS   Medoh UN, Hims A, Chen JY, Ghoochani A, Nyame K, Dong W, 
            Abu-Remaileh M
  TITLE     The Batten disease gene product CLN5 is the lysosomal 
            bis(monoacylglycero)phosphate synthase.
  JOURNAL   Science. 2023 Sep 15;381(6663):1182-1189. doi: 
            10.1126/science.adg9288.
  PUBMED    37708259
REFERENCE   2  (bases 1 to 5771)
  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: "10.1126/science"; 
            journalName: "Science"; date: "2023-09-15- 15"; volume: "381"; 
            issue: "6663"; pages: "1182-1189"
COMMENT     Sequence Label: pRK5 BMPS K319E-6xHis
FEATURES             Location/Qualifiers
     source          1..5771
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     50..72
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     64..81
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     65..81
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(101..458)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      115..250
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        267..458
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     415..435
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    527..661
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(551..570)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     605..624
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     regulatory      665..674
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(711..728)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1867..1886)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        complement(1899..1917)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1900..1917)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2127..2151)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(2148..2351)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2177..2197)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2352..2731
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(2760..2776)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2760..2776)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2773..2795)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2784..2800
                     /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(2808..2814,2815..2832,2833..2838))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2853..2874
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2991..3008)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3162..3750)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3242..3261)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3921..4712,4713..4781))
                     /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     4544..4563
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4782..4886)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4954..4972
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5010..5032)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5132..5151
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5168..5623)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5305..5326)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5517..5536
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
       61 cgttgtaaaa cgacggccag tgccaagctg ttaacagctt tttgcaaaag cctaggcctc
      121 caaaaaagcc tcctcactac ttctggaata gctcagaggc cgaggcggcc tcggcctctg
      181 cataaataaa aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta
      241 ggggcgggat gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc
      301 tttgcatact tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat
      361 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta
      421 actgacacac attccacagc tggttctttc cgcctcagaa ggtacctaac caagttcctc
      481 tttcagaggt tatttcaggc catggtgctg cgccgaatta attcccgatc cagacatgat
      541 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
      601 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
      661 tgggccatgg cggccaagct ggggatcctt acttacttag cggccgctta gtgatggtgg
      721 tgatggtggc ttccgccgcc cccgaggcct gacagagtct tgttcctaat ggggaggggg
      781 atctcctcgt atgtgatctt gatgaaaggg aatttcatgg ggaggaacca atattcgaaa
      841 ttgtaaaaca agtaaaactg ttcatggacg atcacagcat caaaaatctg caggagactc
      901 aggaggaact ccttggtggg caaatgtggc ttgaaagggt agtagaagcg tttgatggcc
      961 aaaccgagcg tcttattccc ggtaggaccg aacactgagg tttcgttgcc caggtaagtg
     1021 ggctcgccgg aataaagaaa gatcctggtg taattggttt caatattttt gaattcggct
     1081 ccgaactctg ccagcttatt aaaagttctc aggacaaatt tgctgcaatc ataactgtcg
     1141 aaccaagttt cagcgccctt ctcaggtgat gccttcacgt tccatgtttc gtagtatatg
     1201 cctgtctcgt tgtcctgctt cacccatttg gccatctggt taaacatatt gccgctaatt
     1261 gtcgcgacct gtaccagggt gccattctct ttccaatgca cgtcgtcgat cccttcgaag
     1321 aagcaggcag cgccctggtt acaccagaat ggtgcatcca tctcaggtct cagatgtggg
     1381 aatgtgcaat tgcccagctg gaagagttca taccactcca tggtgtaatt ctttcctgtg
     1441 agggtagacc ggaaacctat tgcgtcatgc atgattttca gatggccaag caggtcaccg
     1501 tacttgaact cccacacggg ggcttgcaat ctaaacactt cgatgtcatc gtctccctcc
     1561 atgacgggta tgggtgaccc agtagggcaa aatgtgtact tggcctggca atacgggtcg
     1621 ggctttggtc tgaagtcaaa tctcttataa ggaactggcc agtgtcggcg agatgggatg
     1681 cctgaaactc tgctccaccc aggcacgaca gccagccaca ggagggcgag tgcccaacac
     1741 caggaggccc gtcctctggc ggccccggcg ccccgtctca tctctgctcc ctgtgctgtg
     1801 tcgacctcct gggccatggt gaattcaatc gatagaaccg aggtgcagtt ggacctggga
     1861 gtggacacct gtggagagaa aggcaaagtg gatgttattc tatagtgtca cctaaatcgt
     1921 atgtgtatga tacataaggt tatgtattaa ttgtagccgc gttctaacga agccaagggg
     1981 gtgggcctat agactctata ggcggtactt acgtcactct tggcacgggg aatccgcgtt
     2041 ccaatgcacc gttcccggcc gcggaggctg gatcggtccc ggtgtcttct atggaggtca
     2101 aaacagcgtg gatggcgtct ccaggcgatc tgacggttca ctaaacgagc tctgcttata
     2161 tagacctccc accgtacacg cctaccgccc atttgcgtca atggggcgga gttgttacga
     2221 cattttggaa agtcccgttg attttggtgc caaaacaaac tcccattgac gtcaatgggg
     2281 tggagacttg gaaatccccg tgagtcaaac cgctatccac gcccattgat gtactgccaa
     2341 aaccgcatca ccatggtaat agcgatgact aatacgtaga tgtactgcca agtaggaaag
     2401 tcccataagg tcatgtactg ggcataatgc caggcgggcc atttaccgtc attgacgtca
     2461 atagggggcg tacttggcat atgatacact tgatgtactg ccaagtgggc agtttaccgt
     2521 aaatactcca cccattgacg tcaatggaaa gtccctattg gcgttactat gggaacatac
     2581 gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc agccaggcgg gccatttacc
     2641 gtaagttatg taacgcggaa ctccatatat gggctatgaa ctaatgaccc cgtaattgat
     2701 tactattaat aactagtcaa taatcaatgt cgggcgagct cgaattaatt cgtaatcatg
     2761 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     2821 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     2881 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     2941 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     3001 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3061 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     3121 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     3181 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     3241 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     3301 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     3361 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     3421 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     3481 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     3541 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     3601 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     3661 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     3721 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     3781 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     3841 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     3901 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     3961 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     4021 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     4081 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     4141 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     4201 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     4261 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     4321 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     4381 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     4441 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     4501 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     4561 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     4621 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     4681 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     4741 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     4801 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     4861 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     4921 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     4981 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     5041 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     5101 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
     5161 caccataaaa ttgtaaacgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     5221 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     5281 cccgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     5341 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     5401 tcacccaaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     5461 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     5521 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     5581 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtactatgg ttgctttgac
     5641 gtatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgccatt
     5701 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     5761 gccagctggc g
//
