LOCUS       sequence-424658-        6464 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PLBD2-6xHis in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6464)
  AUTHORS   Nyame K, Hims A, Aburous A, Laqtom NN, Dong W, Medoh UN, Heiby JC, 
            Xiong J, Ori A, Abu-Remaileh M
  TITLE     Glycerophosphodiesters inhibit lysosomal phospholipid catabolism in 
            Batten disease.
  JOURNAL   Mol Cell. 2024 Apr 4;84(7):1354-1364.e9. doi: 
            10.1016/j.molcel.2024.02.006. Epub 2024 Mar 5.
  PUBMED    38447580
REFERENCE   2  (bases 1 to 6464)
  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.2024.02.006"; journalName: "Mol Cell"; date: 
            "2024-04-4- 4"; volume: "84"; issue: "7"; pages: "1354-1364.e9"
COMMENT     Sequence Label: pRK5-PLBD2-6xHis
FEATURES             Location/Qualifiers
     source          1..6464
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        828..846
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     828..845
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     859..878
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             2710..2727
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      2764..2773
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2777..2911
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2814..2833)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2868..2887
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2980..3337
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2980..3171
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3003..3023)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3188..3323
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3357..3374)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3357..3373)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3366..3388)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3586..4041
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3673..3692)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3883..3904
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4058..4077)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4177..4199
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4237..4255)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4323..4427
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4428..4496,4497..5288)
                     /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     complement(4646..4665)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5459..6047
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5948..5967
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6201..6218
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6335..6356
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6371..6376,6377..6394,6395..6401)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6409..6425
                     /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     6414..6436
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6433..6449
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6433..6449
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
      721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
      781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
      841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc
      901 acctcggttc tatcgattga attcaccatg gtgggccaga tgtactgcta tccaggatct
      961 catttggcta gagctcttac aagggctctt gcattggctt tagtcttagc attgctcgta
     1021 ggaccctttc tctccggtct tgcaggagca atacctgctc caggcggaag gtgggcacga
     1081 gatgggcagg tccctccagc ctcccgcagc cgctcggtgc tcctggacgt ctcggcgggc
     1141 cagctgctta tggtggacgg acgccaccct gacgccgtgg cctgggccaa cctcaccaac
     1201 gccatccgcg agactgggtg ggccttcctg gagctgggca caagtggcca atacaatgac
     1261 agcttgcagg cctatgcagc cggtgtggtg gaggctgctg tgtcggagga gctcatctac
     1321 atgcactgga tgaacacggt ggtgaattac tgcggcccct tcgagtatga agtcggctac
     1381 tgcgagaggc tgaagagctt cctggaggcc aacctagagt ggatgcagga agagatggag
     1441 tcaaacccag actcacctta ctggcaccag gtgcggctga ccctcctgca gctgaaaggc
     1501 ctggaggaca gctacgaagg ccgtgtgagc ttcccagctg ggaagttcac catcaaaccc
     1561 ttggggttcc tcctgctgca gctctctggg gacctggaag acctggagct ggccctgaac
     1621 aagaccaaga tcaaaccttc tctgggctct ggctcctgtt ctgccctcat caagctgctc
     1681 cctggccaga gtgacctcct ggttgcccac aacacctgga acaactacca gcacatgctg
     1741 cgtgtcatca agaagtactg gctccagttc cgggaaggcc cctgggggga ctacccgctg
     1801 gttcccggca acaagctggt cttctcctcc taccccggca ccatcttctc ctgcgacgac
     1861 ttctacatcc tgggcagtgg gctggtgaca ctggagacca ccattggcaa caagaaccca
     1921 gccctgtgga agtatgtgcg gcccaggggc tgtgtgctgg agtgggtacg caacatcgtg
     1981 gccaaccgcc tggcctcgga tggggccacc tgggcagaca tcttcaagag gttcaacagc
     2041 ggcacgtata acaaccagtg gatgatcgtg gactacaagg cgttcatccc gggtgggccc
     2101 agccccggga gccgggtgct taccatcctg gagcagatcc ccggcatggt ggtggtggct
     2161 gacaagacct cggagctcta ccagaagacc tactgggcca gctacaacat accgtccttc
     2221 gagactgtgt tcaatgccag tgggctgcag gccctagtgg cccagtatgg ggactggttt
     2281 tcttatgacg ggagcccccg ggcccagatc ttccggcgga accagtcact ggtacaagac
     2341 atggactcca tggtcaggct gatgaggtac aatgacttcc tccatgaccc tctgtcactg
     2401 tgcaaagcct gcaaccccca gcccaatggg gagaatgcta tctccgcccg ctccgacctc
     2461 aacccggcca atggctccta ccccttccag gccctgcgtc agcgctccca tgggggtatc
     2521 gatgtgaagg tgaccagcat gtcactggcc aggatcctga gcctgctggc ggccagcggt
     2581 cccacgtggg accaggtgcc cccgttccag tggagcacct cgcccttcag cggcctgctg
     2641 cacatgggcc agccagacct ctggaagttc gcgcctgtca aggtttcatg ggacggaggt
     2701 ggaggtagtc atcatcacca tcaccactag gcggccgcta agtaagtaag gatccccagc
     2761 ttggccgcca tggcccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     2821 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     2881 actcatcaat gtatcttatc atgtctggat cgggaattaa ttcggcgcag caccatggcc
     2941 tgaaataacc tctgaaagag gaacttggtt aggtaccttc tgaggcggaa agaaccagct
     3001 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3061 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     3121 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     3181 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     3241 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     3301 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctgttaacag cttggcactg
     3361 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     3421 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     3481 tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg
     3541 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     3601 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3661 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3721 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3781 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     3841 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     3901 ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg attttgccga
     3961 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     4021 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     4081 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4141 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4201 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     4261 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     4321 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4381 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     4441 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     4501 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     4561 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     4621 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     4681 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     4741 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     4801 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     4861 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     4921 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     4981 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     5041 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     5101 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     5161 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     5221 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     5281 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     5341 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     5401 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     5461 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     5521 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     5581 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     5641 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     5701 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     5761 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     5821 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     5881 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     5941 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     6001 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     6061 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     6121 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     6181 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     6241 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     6301 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     6361 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     6421 aacaatttca cacaggaaac agctatgaca tgattacgaa ttaa
//
