LOCUS       sequence-368095-        7678 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of recombinant kindlin in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7678)
  AUTHORS   Case LB, De Pasquale M, Henry L, Rosen MK
  TITLE     Synergistic phase separation of two pathways promotes integrin 
            clustering and nascent adhesion formation.
  JOURNAL   Elife. 2022 Jan 20;11. pii: 72588. doi: 10.7554/eLife.72588.
  PUBMED    35049497
REFERENCE   2  (bases 1 to 7678)
  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: "Elife."; 
            date: "2022-01-20"; volume: "11. pii: 72588. doi"; pages: " 
            10.7554/eLife.72588"
COMMENT     Sequence Label: Kindlin
FEATURES             Location/Qualifiers
     source          1..7678
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     964..983
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1003..1080)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1286..1305
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1389..1408
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1389..1407
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1408..1432
                     /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             1447..1469
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1488..1505
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1515..1532
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1545..1838
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             3931..3948
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4001..4019)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4015..4062
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4099..4554
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4186..4205)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4396..4417
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4646..5461)
                     /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             complement(4646..5461)
                     /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     5369..5388
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5453..5472
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5583..6171
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6072..6091
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6325..6342
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6357..6496
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6422..6441)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6582..6604
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6598..6789)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7598..7678,1..1002))
                     /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"
ORIGIN
        1 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
       61 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
      121 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
      181 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
      241 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
      301 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
      361 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
      421 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
      481 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
      541 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
      601 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
      661 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
      721 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
      781 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
      841 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
      901 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
      961 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     1021 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     1081 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     1141 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     1201 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     1261 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     1321 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
     1381 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     1441 aataattttg tttaacttta agaaggagat ataccatggg cagcagccat catcatcatc
     1501 atcacagcag cggcctggtg ccgcgcggca gccatatggc tagcatgtcg gactcagaag
     1561 tcaatcaaga agctaagcca gaggtcaagc cagaagtcaa gcctgagact cacatcaatt
     1621 taaaggtgtc cgatggatct tcagagatct tcttcaagat caaaaagacc actcctttaa
     1681 gaaggctgat ggaagcgttc gctaaaagac agggtaagga aatggactcc ttaagattct
     1741 tgtacgacgg tattagaatc caagctgatc agacccctga agatttggac atggaggata
     1801 acgatattat tgaggctcac agagaacaga ttggtggatc catggctctg gacgggataa
     1861 ggatgccaga tggctgctac gcggacggga cgtgggaact gagtgtccat gtgacggacc
     1921 tgaaccgcga tgtcaccctg agagtgaccg gcgaggtgca cattggaggc gtgatgctta
     1981 agctggtgga gaaactcgat gtaaaaaaag attggtctga ccatgctctc tggtgggaaa
     2041 agaagagaac ttggcttctg aagacacatt ggaccttaga taagtatggt attcaggcag
     2101 atgctaagct tcagttcacc cctcagcaca aactgctccg cctgcagctt cccaacatga
     2161 agtatgtgaa ggtgaaagtg aatttctctg atagagtctt caaagctgtt tctgacatct
     2221 gtaagacttt taatatcaga caccccgaag aactttctct cttaaagaaa cccagagatc
     2281 caacaaagaa aaaaaagaag aagctagatg accagtctga agatgaggca cttgaattag
     2341 aggggcctct tatcactcct ggatcaggaa gtatatattc aagcccagga ctgtatagta
     2401 aaacaatgac ccccacttat gatgctcatg atggaagccc cttgtcacca acttctgctt
     2461 ggtttggtga cagtgctttg tcagaaggca atcctggtat acttgctgtc agtcaaccaa
     2521 tcacgtcacc agaaatcttg gcaaaaatgt tcaagcctca agctcttctt gataaagcaa
     2581 aaatcaacca aggatggctt gattcctcaa gatctctcat ggaacaagat gtgaaggaaa
     2641 atgaggcctt gctgctccga ttcaagtatt acagcttttt tgatttgaat ccaaagtatg
     2701 atgcaatcag aatcaatcag ctttatgagc aggccaaatg ggccattctc ctggaagaga
     2761 ttgaatgcac agaagaagaa atgatgatgt ttgcagccct gcagtatcat atcaataagc
     2821 tgtcaatcat gacatcagag aatcatttga acaacagtga caaagaagtt gatgaagttg
     2881 atgctgccct ttcagacctg gagattactc tggaaggggg taaaacgtca acaattttgg
     2941 gtgacattac ttccattcct gaacttgctg actacattaa agttttcaag ccaaaaaagc
     3001 tgactctgaa aggttacaaa caatattggt gcaccttcaa agacacatcc atttcttgtt
     3061 ataagagcaa agaagaatcc agtggcacac cagctcatca gatgaacctc aggggatgtg
     3121 aagttacccc agatgtaaac atttcaggcc aaaaatttaa cattaaactc ctgattccag
     3181 ttgcagaagg catgaatgaa atctggcttc gttgtgacaa tgaaaaacag tatgcacact
     3241 ggatggcagc ctgcagatta gcctccaaag gcaagaccat ggcggacagt tcttacaact
     3301 tagaagttca gaatattctt tcctttctga agatgcagca tttaaaccca gatcctcagt
     3361 taataccaga gcagatcacg actgatataa ctcctgaatg tttggtgtct ccccgctatc
     3421 taaaaaagta taagaacaag cagataacag cgagaatctt ggaggcccat cagaatgtag
     3481 ctcagatgag tctaattgaa gccaagatga gatttattca agcttggcag tcactacctg
     3541 aatttggcat cactcacttc attgcaaggt tccaaggggg caaaaaagaa gaacttattg
     3601 gaattgcata caacagactg attcggatgg atgccagcac tggagatgca attaaaacat
     3661 ggcgtttcag caacatgaaa cagtggaatg tcaactggga aatcaaaatg gtcaccgtag
     3721 agtttgcaga tgaagtacga ttgtccttca tttgtactga agtagattgc aaagtggttc
     3781 atgagttcat tggtggctac atatttctct caacacgtgc aaaagaccaa aacgagagtt
     3841 tagatgaaga gatgttctac aaacttacca gtggttgggt gtagtaataa gaattcgagc
     3901 tccgtcgaca agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct
     3961 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     4021 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     4081 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4141 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     4201 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     4261 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     4321 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     4381 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     4441 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     4501 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     4561 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     4621 tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca
     4681 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     4741 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     4801 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     4861 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt
     4921 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     4981 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa
     5041 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     5101 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga
     5161 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     5221 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     5281 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     5341 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     5401 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca
     5461 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc
     5521 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5581 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     5641 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     5701 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     5761 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     5821 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     5881 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     5941 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6001 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6061 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6121 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6181 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     6241 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     6301 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     6361 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     6421 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     6481 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     6541 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     6601 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     6661 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     6721 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     6781 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     6841 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     6901 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     6961 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     7021 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     7081 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     7141 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     7201 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc
     7261 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     7321 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     7381 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     7441 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     7501 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     7561 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     7621 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttg
//
