LOCUS       sequence-443086-        5524 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the 4-helix bundle of TLNRD1 (residues 
            143-273) containing the mutations L191T/A225T, fused to an 
            N-terminal hexa-histidine tag. HRV-3C cleavage site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5524)
  AUTHORS   Ball NJ, Ghimire S, Follain G, Pajari AO, Wurzinger D, Vaitkeviciute
            M, Cowell AR, Berki B, Ivaska J, Paatero I, Goult BT, Jacquemet G
  TITLE     TLNRD1 is a CCM complex component and regulates endothelial barrier 
            integrity.
  JOURNAL   J Cell Biol. 2024 Sep 2;223(9):e202310030. doi: 
            10.1083/jcb.202310030. Epub 2024 Jul 16.
  PUBMED    39013281
REFERENCE   2  (bases 1 to 5524)
  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: "J Cell 
            Biol."; date: "2024-09-2"; volume: "223(9):e202310030. doi"; pages: 
            " 10.1083/jcb.202310030. Epub 2024 Jul 16"
COMMENT     Sequence Label: TLNRD1(4H)-2T_pET47b
FEATURES             Location/Qualifiers
     source          1..5524
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(168..212)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(213..230)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(634..657)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(667..684)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             698..720
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    735..759
                     /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     complement(759..778)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(760..778)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(866..885)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1091..1168
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1169..2251
                     /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(1188..1207)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2823..3014
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3008..3030)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3116..3255
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3171..3190
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3270..3287)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3441..4029)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3521..3540)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4140..4159)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4151..4966
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4151..4966
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4224..4243)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5058..5513)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5195..5216)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5407..5426
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgtttagcag cctaggttaa ttaagcctcg agagcagcag aagtagagct gtccatgtgc
      181 tggcgttcga atttagcagc agcggtttct ttactaccgc gtggcaccag agcgttaacg
      241 acccagaaac tgcggttcgg ttgcaaaacc aaccagtgcg ctaactgcct gaaccagcgg
      301 accgctaaac agtgcacaac ggctacgtgc cagttcgctc ggtgcaactt taacttcacg
      361 aacacatgcc agcagtgcgc tggtgctggt gctcatacat ttaacaccca gtttgaactg
      421 ttcacggcta aaacgatcac ggcttttatc gcttgccagt gcacatgcat cggtcagaaa
      481 tttggtatta cggctcagac cctggctaac ttctaacagc agctgcggtg tcatatctgc
      541 cagcggtgtg gcacgcagaa ctgcacaacc ctgttcaact tcatgacgac aacgggtaac
      601 acgataacga tcaaccagac caggctgtgc accgggtccc tgaaagagga cttcaagagc
      661 cgcggagtga tggtggtggt gatgtgccat atgtatatct ccttcttaaa gttaaacaaa
      721 attatttcta gaggggaatt gttatccgct cacaattccc ctatagtgag tcgtattaat
      781 ttcgcgggat cgagatcgat ctcgatcctc tacgccggac gcatcgtggc cggcatcacc
      841 ggcgccacag gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg
      901 gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg
      961 gccgggggac tgttgggcgc catctccttg catgcaccat tccttgcggc ggcggtgctc
     1021 aacggcctca acctactact gggctgcttc ctaatgcagg agtcgcataa gggagagcgt
     1081 cgagatcccg gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc
     1141 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
     1201 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
     1261 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
     1321 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
     1381 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     1441 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     1501 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     1561 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     1621 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
     1681 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     1741 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
     1801 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
     1861 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
     1921 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggaca tctcggtagt
     1981 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
     2041 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     2101 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
     2161 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     2221 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca
     2281 ctcattaggc accgggatct cgaccgatgc ccttgagagc cttcaaccca gtcagctcct
     2341 tccggtgggc gcggggcatg actagcatga tcgtgctcct gtcgttgagg acccggctag
     2401 gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa
     2461 gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc
     2521 gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct
     2581 gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct
     2641 ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac
     2701 cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc
     2761 tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat
     2821 cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat
     2881 taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat
     2941 cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg
     3001 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     3061 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag
     3121 ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga
     3181 gcagattgta ctgagagtgc accattgcgg tgtgaaatac cgcacagatg cgtaaggaga
     3241 aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     3301 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     3361 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     3421 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     3481 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     3541 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     3601 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     3661 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     3721 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     3781 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     3841 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     3901 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     3961 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4021 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4081 tcacgttaag ggattttggt catgaacaat aaaactgtct gcttacataa acagtaatac
     4141 aaggggtgtt atgagccata ttcaacggga aacgtcttgc tctaggccgc gattaaattc
     4201 caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg
     4261 tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg
     4321 caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga
     4381 atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact
     4441 caccactgcg atccccggca aaacagcatt ccaggtatta gaagaatatc ctgattcagg
     4501 tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg
     4561 taattgtcct tttaacagtg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa
     4621 taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca
     4681 agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg
     4741 tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt
     4801 tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg
     4861 tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga
     4921 tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaagaat taattcatga
     4981 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5041 cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     5101 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     5161 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     5221 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     5281 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     5341 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     5401 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     5461 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc
     5521 gcca
//
