LOCUS       sequence-443085-        6153 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the 4-helix bundle of TLNRD1 (residues 
            143-273) containing the mutations K192E/R233E, fused to an 
            N-terminal hexa-histidine tag. TEV cleavage site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6153)
  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 6153)
  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)-2E_pET151
FEATURES             Location/Qualifiers
     source          1..6153
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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)."
     RBS             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             300..317
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             318..359
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             360..380
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(854..872)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      868..915
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1153..1171)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1239..1343
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1344..1412,1413..2204)
                     /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(1562..1581)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2375..2963
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2864..2883
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3117..3134
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3149..3288
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3214..3233)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3374..3396
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3390..3581)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4893..5975)
                     /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     5937..5956
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5976..6053)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatgc
      301 atcatcacca tcaccatggt aagcctatcc ctaaccctct cctcggtctc gattctacgg
      361 aaaacctgta ttttcaggga attgatccct tcaccgcaca gcctggtctg gttgatcgtt
      421 atcgtgttac ccgttgtcgt catgaagttg aacagggttg tgcagttctg cgtgcaacac
      481 cgctggcaga tatgacaccg cagctgctgc tggaagttag ccagggtctg agccgtaatc
      541 tggaatttct gaccgatgca tgtgcactgg caagcgataa aagccgtgat cgttttagcc
      601 gtgaacagtt caaactgggt gttaaatgta tgagcaccag cgcaagcgca ctgctggcat
      661 gtgttgaaga agttaaagtt gcaccgagcg aactggcacg tagccgttgt gcactgttta
      721 gcggtccgct ggttcaggca gttagcgcac tggttggttt tgcaaccgaa ccgcagtttc
      781 tgggtcgtta aaagggcgag ctcagatccg gctgctaaca aagcccgaaa ggaagctgag
      841 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
      901 ttgaggggtt ttttgctgaa aggaggaact atatccggat atcccgcaag aggcccggca
      961 gtaccggcat aaccaagcct atgcctacag catccagggt gacggtgccg aggatgacga
     1021 tgagcgcatt gttagatttc atacacggtg cctgactgcg ttagcaattt aactgtgata
     1081 aactaccgca ttaaagctag cttatcgatg ataagctgtc aaacatgaga attaattctt
     1141 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     1201 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     1261 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     1321 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     1381 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     1441 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     1501 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     1561 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     1621 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     1681 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     1741 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     1801 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     1861 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     1921 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     1981 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     2041 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     2101 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     2161 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     2221 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     2281 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     2341 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     2401 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     2461 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     2521 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     2581 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     2641 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     2701 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     2761 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     2821 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     2881 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     2941 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     3001 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     3061 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     3121 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     3181 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     3241 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     3301 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     3361 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     3421 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     3481 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     3541 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     3601 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     3661 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     3721 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     3781 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     3841 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     3901 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     3961 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     4021 gacaggagca cgatcatgcg cacccgtggc caggacccaa cgctgcccga gatgcgccgc
     4081 gtgcggctgc tggagatggc ggacgcgatg gatatgttct gccaagggtt ggtttgcgca
     4141 ttcacagttc tccgcaagaa ttgattggct ccaattcttg gagtggtgaa tccgttagcg
     4201 aggtgccgcc ggcttccatt caggtcgagg tggcccggct ccatgcaccg cgacgcaacg
     4261 cggggaggca gacaaggtat agggcggcgc ctacaatcca tgccaacccg ttccatgtgc
     4321 tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc aatgatcgaa gttaggctgg
     4381 taagagccgc gagcgatcct tgaagctgtc cctgatggtc gtcatctacc tgcctggaca
     4441 gcatggcctg caacgcgggc atcccgatgc cgccggaagc gagaagaatc ataatgggga
     4501 aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta gcccagcgcg tcggccgcca
     4561 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     4621 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     4681 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     4741 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     4801 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     4861 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4921 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4981 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     5041 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     5101 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     5161 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     5221 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     5281 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     5341 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     5401 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     5461 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     5521 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     5581 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     5641 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     5701 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     5761 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     5821 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     5881 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     5941 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     6001 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     6061 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     6121 gttgagcacc gccgccgcaa ggaatggtgc atg
//
