LOCUS       sequence-461397-        8388 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Human RLIM N581K .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8388)
  AUTHORS   Bandi V, Rennie M, Koch I, Gill P, Pacheco OD, Berg AD, Cui H, Ward 
            DI, Bustos F
  TITLE     RLIM-specific activity reporters define variant pathogenicity in 
            Tonne-Kalscheuer syndrome.
  JOURNAL   HGG Adv. 2024 Oct 31;6(1):100378. doi: 10.1016/j.xhgg.2024.100378.
  PUBMED    39482882
REFERENCE   2  (bases 1 to 8388)
  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: "HGG Adv."; 
            date: "2024-10-31"; volume: "6(1):100378. doi"; pages: " 
            10.1016/j.xhgg.2024.100378"
COMMENT     Sequence Label: pCAGGS puro human RLIM N581K
FEATURES             Location/Qualifiers
     source          1..8388
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     14..33
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(252..356)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      383..838
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(470..489)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     680..701
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     964..986
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     978..995
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     979..995
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(1331..1930)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1414..1434)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     1911..1930
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(1946..2445)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1994..2013)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     2387..2410
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     polyA_signal    2450..2584
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2474..2493)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2528..2547
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     regulatory      4816..4825
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4860..4879)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          4887..5903
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(4941..4963)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(5904..6179)
                     /label=chicken beta-actin promoter
     enhancer        6181..6560
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(6569..6588)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6570..6588)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6618..6634)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6618..6634)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6631..6653)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6642..6658
                     /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(6666..6672,6673..6690,6691..6696))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6711..6732
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6849..6866)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7020..7608)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7100..7119)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7779..8388,1..182,183..251))
                     /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"
ORIGIN
        1 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
       61 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      121 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      181 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
      241 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
      301 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
      361 tttccccgaa aagtgccacc tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
      421 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
      481 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
      541 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
      601 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
      661 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
      721 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
      781 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttcc
      841 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      901 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      961 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gctgacttgg tcagcggccg
     1021 gccgcgtcga gccccagctg gttctttccg cctcagaagc catagagccc accgcatccc
     1081 cagcatgcct gctattgtct tcccaatcct cccccttgct gtcctgcccc accccacccc
     1141 ccagaataga atgacaccta ctcagacaat gcgatgcaat ttcctcattt tattaggaaa
     1201 ggacagtggg agtggcacct tccagggtca aggaaggcac gggggagggg caaacaacag
     1261 atggctggca aatcgatgca tggggtcgtg cgctcctttc ggtcgggcgc tgcgggtcgt
     1321 ggggcgggcg tcaggcaccg ggcttgcggg tcatgcacca ggtgcgcggt ccttcgggca
     1381 cctcgacgtc ggcggtgacg gtgaagccga gccgctcgta gaaggggagg ttgcggggcg
     1441 cggaggtctc caggaaggcg ggcaccccgg cgcgctcggc cgcctccact ccggggagca
     1501 cgacggcgct gcccagaccc ttgccctggt ggtcgggcga gacgccgacg gtggccagga
     1561 accacgcggg ctccttgggc cggtgcggcg ccaggaggcc ttccatctgt tgctgcgcgg
     1621 ccagccggga accgctcaac tcggccatgc gcgggccgat ctcggcgaac accgcccccg
     1681 cttcgacgct ctccggcgtg gtccagaccg ccaccgcggc gccgtcgtcc gcgacccaca
     1741 ccttgccgat gtcgagcccg acgcgcgtga ggaagagttc ttgcagctcg gtgacccgct
     1801 cgatgtggcg gtccggatcg acggtgtggc gcgtggcggg gtagtcggcg aacgcggcgg
     1861 cgagggtgcg tacggccctg gggacgtcgt cgcgggtggc gaggcgcacc gtgggcttgt
     1921 actcggtcat ggtaagctag cttgtcgaaa ggcccggaga tgaggaagag gagaacagcg
     1981 cggcagacgt gcgcttttga agcgtgcaga atgccgggcc tccggaggac cttcgggcgc
     2041 ccgccccgcc cctgagcccg cccctgagcc cgcccccgga cccacccctt cccagcctct
     2101 gagcccagaa agcgaaggag caaagctgct attggccgct gccccaaagg cctacccgct
     2161 tccattgctc agcggtgctg tccatctgca cgagactagt gagacgtgct acttccattt
     2221 gtcacgtcct gcacgacgcg agctgcgggg cgggggggaa cttcctgact aggggaggag
     2281 tagaaggtgg cgcgaagggg ccaccaaaga acggagccgg ttggcgccta ccggtggatg
     2341 tggaatgtgt gcgaggccag aggccacttg tgtagcgcca agtgcccagc ggggctgcta
     2401 aagcgcatgc tccagactgc cttgggaaaa gcgcctcccc tacccggtag atccagacat
     2461 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     2521 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     2581 agttccgagt ttgtcagaaa gcagaccaaa cagcggttgg aataatagcg agaacagaga
     2641 aatagcggca aaaataatac ccgtatcact tttgctgata tggttgatgt catgtagcca
     2701 aatcgggaaa aacgggaagt aggctcccat gataaaaaag taaaagaaaa agaataaacc
     2761 gaacatccaa aagtttgtgt tttttaaata gtacataatg gatttcctta cgcgaaatac
     2821 gggcagacat ggcctgcccg gttattatta tttttgacac cagaccaact ggtaatggta
     2881 gcgaccggcg ctcagctgga attggcggcc gcatgcattt gctagccctt aattaaggct
     2941 cgagttacac aacactttct ctgttaccag aagctaagac tgctctgcga caaataggac
     3001 aggtagaatt ctcagataac cagcgatcga tgcagtggac atggtactca tgggaacaag
     3061 gtagtttacg aagtttcttg ccttctgtat attctgtaat gcaaacacta caggttttta
     3121 atgcatcatt ttcaccaaaa cttctcattg ccaagttgtc aatctgttct ttggtgagtc
     3181 ctctaggttg gtcatcatca tcctcattta agaggaaaaa ctgagccagg ctaaggaagg
     3241 gcaaagagcc actttcatca aatgtgactg gggccctatg tcgaccctct cgcctggcac
     3301 ctgatgagcc tgatgatgag cttccttcat tactgccttc aaataaatct gagctagttt
     3361 ctgaactttc accaccggaa ctggaactag gactggaact ggaacttgaa ctggaactgg
     3421 aactcgaact ggaactggaa ctcgaactgg aaccagaact actaccacca ccagaacctc
     3481 ctcttccact ccgtgactct gccctttcca tatttcgatt tgagactgag ccagtaggct
     3541 ctgagtcgct atcactgtac ataaaatagc ttaactcacc aaaacctgtc attatctgcc
     3601 ttaacatggt ctgaattgca acagatgtag tctcacttaa accagtattt aagattctac
     3661 gaatgggaat tctgatggta ctgacatagg ttctcacacc tgcccgctca gaacgtgaaa
     3721 atgtacgcct aaaacctcct cgttcacttt cataggtgac agtgttgttt ggtgtctgag
     3781 acctagaccg agttctgctg gctatgctat ctctctgccg atattctcca ggacgaactc
     3841 ttcttacttg aagatcaagg actatggttg gaggtctctg tcctgatcct gtagattcac
     3901 cactggcagc tgtgtctgaa gaacctgctc cttgagaagc atttcttgtt ccagaagctg
     3961 caaaaagacc tctacttagc aactcaggcc cagatatttg ctgcctcaat gtcacatggt
     4021 gccgggttct agaacttccc tccgtctcat ttaccaaagg atgttcaaaa gtctgagatg
     4081 agatactatg atgagatctt cgtggaattt cactcattgg atgcagaggt gacctacttc
     4141 tttcagctct tgctctggtt ctccgatggt ctgggctcct gcttcttgcc ctcctctgac
     4201 ctctggtagg tgggacctct gttaacgctt cagttgaatt tcgttctgat ctagatggcc
     4261 ttgcagatgt tgattcagat cgtgggtttt ccacttgcct ttggctgttg ttttccacat
     4321 tttctccact agaacgtctt gcagatggct cattttcatt ctctgaattt tggctcccat
     4381 tattacggtt aacatttatc tctaaactga atctgaaatc accactgttt ggattagtcc
     4441 gactcactgc tctccaagat tggtttcctc tttgcccact tcttgttgta tttccagttt
     4501 gtctgacaga gttaagccag tctattatag agtcaccatt agacacatca tctgaagagt
     4561 ctcctcctct attttcatct gagttttgcg gtggtgggcc ttctttaatt tgctgtagtc
     4621 gtctcagcaa ctcttcctca gtactttcac ctggggtgcc tagcaaattg ttatctctca
     4681 taagcctata atcttcttca ctcaggttat ttacaaattg atagaaagct tcttctcgat
     4741 ccaatcggtc catctgactt ctgcgctgtg ctgcagactg atcaccactt cctttgtcat
     4801 tggaatctga gttttccatg gtggctcgag ttgaattctt tgccaaaatg atgagacagc
     4861 acaataacca gcacgttgcc caggagctgt aggaaaaaga agaaggcatg aacatggtta
     4921 gcagaggctc tagagccgcc ggtcacacgc cagaagccga accccgccct gccccgtccc
     4981 ccccgaaggc agccgtcccc ccgcggacag ccccgaggct ggagagggag aaggggacgg
     5041 cggcgcggcg acgcacgaag gccctccccg cccatttcct tcctgccggc gccgcaccgc
     5101 ttcgccccgc gcccgctaga gggggtgcgg cggcgcctcc cagatttcgg ctccgcacag
     5161 atttgggaca aaggaagtcc ctgcgccctc tcgcacgatt accataaaag gcaatggctg
     5221 cggctcgccg cgcctcgaca gccgccggcg ctccgggggc cgccgcgccc cycccccgag
     5281 cccyccccgg cccgaggcgg ccccgccccg cccggcaccc ccmcctgccg ccaccccccg
     5341 cccggcacgg cgagccccgc gccacgcccc gtacggagcc ccgcacccga agccgggccg
     5401 tgctcagcaa ctcggggagg ggggtgcagg gggggttgca gcccgaccga cgcgcccaca
     5461 ccccctgctc acccccccac gcacacaccc cgcacgcagc ctttgttccc ctcgcagccc
     5521 cccccgcacc gcggggcacc gcccccggcc gcgctcccct cgcgcacact gcggagcgca
     5581 caaagccccg cgccgcgccc gcagcgctca cagccgccgg gcagcgcgga gccgcacgcg
     5641 gcgctcccca cgcacacaca cacgcacgca ccccccgagc cgctcccccc gcacaaaggg
     5701 ccctcccgga gcccctcaag gctttcacgc agccacagaa aagaaacaag ccgtcattaa
     5761 accaagcgct aattacagcc cggaggagaa gggccgtccc gcccgctcac ctgtgggagt
     5821 aacgcggtca gtcagagccg gggcgggcgg cgcgaggcgg cggcggagcg gggcacgggg
     5881 cgaaggcagc gcgcagcgac tcccgcccgc cgcgcgcttc gctttttata gggccgccgc
     5941 cgccgccgcc tcgccataaa aggaaacttt cggagcgcgc cgctctgatt ggctgccgcc
     6001 gcacctctcc gcctcgcccc sccccscccc tcsccccscc ccsccccscc tggcgcgcsc
     6061 cccccccccc cccgccccca tcgctgcaca aaataattaa aaaataaata aatacaaaat
     6121 tgggggtggg gagggggggg agatggggag agtgaagcag aacgtggggc tcacctcgac
     6181 catggtaata gcgatgacta atacgtagat gtactgccaa gtaggaaagt cccataaggt
     6241 catgtactgg gcataatgcc aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt
     6301 acttggcata tgatacactt gatgtactgc caagtgggca gtttaccgta aatactccac
     6361 ccattgacgt caatggaaag tccctattgg cgttactatg ggaacatacg tcattattga
     6421 cgtcaatggg cgggggtcgt tgggcggtca gccaggcggg ccatttaccg taagttatgt
     6481 aacgcggaac tccatatatg ggctatgaac taatgacccc gtaattgatt actattaata
     6541 actagtcaat aatcaatgtc gacgagctcc ctatagtgag tcgtattaga ggccgacttg
     6601 gccaaattcg taatcatgtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     6661 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     6721 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     6781 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     6841 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     6901 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     6961 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     7021 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     7081 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     7141 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     7201 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     7261 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     7321 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     7381 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     7441 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     7501 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     7561 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     7621 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     7681 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     7741 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     7801 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     7861 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     7921 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     7981 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     8041 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     8101 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     8161 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     8221 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     8281 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     8341 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccg
//
