LOCUS       sequence-459790-        6836 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Protein expression of GST-human RLIM N581K .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6836)
  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 6836)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pGEX6P1 human RLIM N581K
FEATURES             Location/Qualifiers
     source          1..6836
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     701..718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        791..868
                     /gene="lacI"
                     /label=lacI promoter
     CDS             869..1951
                     /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     complement(888..907)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(2000..2005,2006..2023,2024..2030)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2038..2054
                     /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     2062..2078
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2062..2078
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2074..2601
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(2094..2111)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2094..2110)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2103..2125)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(2703..2708,2709..2724,2725..2731)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2739..2755
                     /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)."
     CDS             2778..3431
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     3389..3411
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             3438..3461
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(5406..5428)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5573..5591)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5659..5763
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5764..5832,5833..6624)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5982..6001)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6795..6836,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      721 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
      781 cataaattcc gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc
      841 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
      901 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
      961 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
     1021 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
     1081 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     1141 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     1201 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     1261 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     1321 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
     1381 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     1441 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
     1501 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
     1561 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
     1621 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt
     1681 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
     1741 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     1801 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccacgctagc
     1861 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     1921 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca
     1981 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     2041 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgga ttcactggcc
     2101 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     2161 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     2221 caacagttgc gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc accagaagcg
     2281 gtgccggaaa gctggctgga gtgcgatctt cctgaggccg atactgtcgt cgtcccctca
     2341 aactggcaga tgcacggtta cgatgcgccc atctacacca acgtgaccta tcccattacg
     2401 gtcaatccgc cgtttgttcc cacggagaat ccgacgggtt gttactcgct cacatttaat
     2461 gttgatgaaa gctggctaca ggaaggccag acgcgaatta tttttgatgg cgttggaatt
     2521 agcttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
     2581 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
     2641 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
     2701 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
     2761 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
     2821 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
     2881 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
     2941 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
     3001 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
     3061 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
     3121 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
     3181 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
     3241 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
     3301 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
     3361 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
     3421 atcctccaaa atcggatctg gaagttctgt tccaggggcc cctgggatcc atggaaaact
     3481 cagattccaa tgacaaagga agtggtgatc agtctgcagc acagcgcaga agtcagatgg
     3541 accgattgga tcgagaagaa gctttctatc aatttgtaaa taacctgagt gaagaagatt
     3601 ataggcttat gagagataac aatttgctag gcaccccagg tgaaagtact gaggaagagt
     3661 tgctgagacg actacagcaa attaaagaag gcccaccacc gcaaaactca gatgaaaata
     3721 gaggaggaga ctcttcagat gatgtgtcta atggtgactc tataatagac tggcttaact
     3781 ctgtcagaca aactggaaat acaacaagaa gtgggcaaag aggaaaccaa tcttggagag
     3841 cagtgagtcg gactaatcca aacagtggtg atttcagatt cagtttagag ataaatgtta
     3901 accgtaataa tgggagccaa aattcagaga atgaaaatga gccatctgca agacgttcta
     3961 gtggagaaaa tgtggaaaac aacagccaaa ggcaagtgga aaacccacga tctgaatcaa
     4021 catctgcaag gccatctaga tcagaacgaa attcaactga agcgttaaca gaggtcccac
     4081 ctaccagagg tcagaggagg gcaagaagca ggagcccaga ccatcggaga accagagcaa
     4141 gagctgaaag aagtaggtca cctctgcatc caatgagtga aattccacga agatctcatc
     4201 atagtatctc atctcagact tttgaacatc ctttggtaaa tgagacggag ggaagttcta
     4261 gaacccggca ccatgtgaca ttgaggcagc aaatatctgg gcctgagttg ctaagtagag
     4321 gtctttttgc agcttctgga acaagaaatg cttctcaagg agcaggttct tcagacacag
     4381 ctgccagtgg tgaatctaca ggatcaggac agagacctcc aaccatagtc cttgatcttc
     4441 aagtaagaag agttcgtcct ggagaatatc ggcagagaga tagcatagcc agcagaactc
     4501 ggtctaggtc tcagacacca aacaacactg tcacctatga aagtgaacga ggaggtttta
     4561 ggcgtacatt ttcacgttct gagcgggcag gtgtgagaac ctatgtcagt accatcagaa
     4621 ttcccattcg tagaatctta aatactggtt taagtgagac tacatctgtt gcaattcaga
     4681 ccatgttaag gcagataatg acaggttttg gtgagttaag ctattttatg tacagtgata
     4741 gcgactcaga gcctactggc tcagtctcaa atcgaaatat ggaaagggca gagtcacgga
     4801 gtggaagagg aggttctggt ggtggtagta gttctggttc cagttcgagt tccagttcca
     4861 gttcgagttc cagttccagt tcaagttcca gttccagtcc tagttccagt tccggtggtg
     4921 aaagttcaga aactagctca gatttatttg aaggcagtaa tgaaggaagc tcatcatcag
     4981 gctcatcagg tgccaggcga gagggtcgac atagggcccc agtcacattt gatgaaagtg
     5041 gctctttgcc cttccttagc ctggctcagt ttttcctctt aaatgaggat gatgatgacc
     5101 aacctagagg actcaccaaa gaacagattg acaacttggc aatgagaagt tttggtgaaa
     5161 atgatgcatt aaaaacctgt agtgtttgca ttacagaata tacagaaggc aagaaacttc
     5221 gtaaactacc ttgttcccat gagtaccatg tccactgcat cgatcgctgg ttatctgaga
     5281 attctacctg tcctatttgt cgcagagcag tcttagcttc tggtaacaga gaaagtgttg
     5341 tgtaagcggc cgcatcgtga ctgactgacg atctgcctcg cgcgtttcgg tgatgacggt
     5401 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     5461 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
     5521 atgacccagt cacgtagcga tagcggagtg tataattctt gaagacgaaa gggcctcgtg
     5581 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     5641 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     5701 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     5761 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     5821 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     5881 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     5941 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     6001 tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     6061 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     6121 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     6181 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     6241 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     6301 atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     6361 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     6421 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     6481 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     6541 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     6601 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     6661 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     6721 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     6781 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaac
//
