LOCUS       sequence-443082-        5491 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the C-terminal helix of CCM2 (residues 
            413-438) fused to an N-terminal hexa-histidine tag and SUMO(K0) [all
            lysines in SUMO mutated to arginines]. HRV-3C cleavage site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5491)
  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 5491)
  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: CCM2(413-438)_pHisSUMO(K0)
FEATURES             Location/Qualifiers
     source          1..5491
                     /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(319..342)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(634..651)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             665..687
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    702..726
                     /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(726..745)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(727..745)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(833..852)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1058..1135
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1136..2218
                     /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(1155..1174)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2790..2981
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2975..2997)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3083..3222
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3138..3157
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3237..3254)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3408..3996)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3488..3507)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4107..4126)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4118..4933
                     /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             4118..4933
                     /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(4191..4210)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5025..5480)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5162..5183)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5374..5393
                     /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 agcgttagct
      241 acaacccagt gcttcaatat cgctgctaat atcgctaatc atacgatccc attcatcacc
      301 ttcgctcggt gcgctaccgg gtccctgaaa cagaacttcc agaccaccaa tctgctcacg
      361 atgtgcttcg ataatatcgt tatcttccat atccagatct tccggtgtct gatctgcctg
      421 aatacgaata ccatcataca gaaaacgcag gctatccatt tcacgaccct gacgacgtgc
      481 aaatgcttcc atcagacgac gcagcggtgt ggtacgacga atacgaaaaa agatttcgct
      541 gctaccatca ctaacacgca gattaatatg tgtttccgga cgcacttcag gacgaacttc
      601 cggacgtgct tcttgattaa cttcgctatc gctatgatga tggtgatgat gtgccatatg
      661 tatatctcct tcttaaagtt aaacaaaatt atttctagag gggaattgtt atccgctcac
      721 aattccccta tagtgagtcg tattaatttc gcgggatcga gatcgatctc gatcctctac
      781 gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg cgcctatatc
      841 gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag cgcttgtttc
      901 ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat ctccttgcat
      961 gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta
     1021 atgcaggagt cgcataaggg agagcgtcga gatcccggac accatcgaat ggcgcaaaac
     1081 ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa
     1141 accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg
     1201 cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat
     1261 ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt
     1321 gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc
     1381 gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag
     1441 cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct
     1501 gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa
     1561 tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc
     1621 ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat
     1681 cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca
     1741 taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc
     1801 catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat
     1861 gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct
     1921 gcgcgttggt gcggacatct cggtagtggg atacgacgat accgaagaca gctcatgtta
     1981 tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga
     2041 ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc
     2101 actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt
     2161 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     2221 gcaacgcaat taatgtaagt tagctcactc attaggcacc gggatctcga ccgatgccct
     2281 tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact agcatgatcg
     2341 tgctcctgtc gttgaggacc cggctaggct ggcggggttg ccttactggt tagcagaatg
     2401 aatcaccgat acgcgagcga acgtgaagcg actgctgctg caaaacgtct gcgacctgag
     2461 caacaacatg aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag
     2521 cgccctgcac cattatgttc cggatctgca tcgcaggatg ctgctggcta ccctgtggaa
     2581 cacctacatc tgtattaacg aagcgctggc attgaccctg agtgattttt ctctggtccc
     2641 gccgcatcca taccgccagt tgtttaccct cacaacgttc cagtaaccgg gcatgttcat
     2701 catcagtaac ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt acccccatga
     2761 acagaaatcc cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca
     2821 tggcccgctt tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg
     2881 cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca
     2941 gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     3001 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     3061 cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt
     3121 atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc attgcggtgt
     3181 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg
     3241 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     3301 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     3361 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3421 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3481 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     3541 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     3601 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     3661 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     3721 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     3781 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     3841 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     3901 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     3961 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4021 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gaacaataaa
     4081 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac
     4141 gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg
     4201 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga
     4261 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga
     4321 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat
     4381 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccggcaaaa cagcattcca
     4441 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct
     4501 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagtgatc gcgtatttcg
     4561 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga
     4621 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt
     4681 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga
     4741 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga
     4801 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt
     4861 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga
     4921 tgagtttttc taagaattaa ttcatgagcg gatacatatt tgaatgtatt tagaaaaata
     4981 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa
     5041 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     5101 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     5161 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     5221 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     5281 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     5341 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     5401 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     5461 tgcgccgcta cagggcgcgt cccattcgcc a
//
