LOCUS       sequence-365933-        8266 bp    DNA     circular SYN 15-APR-2025
DEFINITION  For expression of recombinant Snap-labeled kindlin in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8266)
  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 8266)
  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: "Elife."; 
            date: "2022-01-20"; volume: "11. pii: 72588. doi"; pages: " 
            10.7554/eLife.72588"
COMMENT     Sequence Label: SNAP-Kindlin
FEATURES             Location/Qualifiers
     source          1..8266
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     956..975
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(995..1072)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1278..1297
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1381..1400
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1381..1399
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1400..1424
                     /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             1439..1461
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1480..1497
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1507..1524
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1537..1830
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             1834..2379
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="optimized for bacterial expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     CDS             4511..4528
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4581..4599)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4595..4642
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4679..5134
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4766..4785)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4976..4997
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5226..6041)
                     /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(5226..6041)
                     /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     5949..5968
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6033..6052
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6163..6751
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6652..6671
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6905..6922
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6937..7076
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7002..7021)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7162..7184
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7178..7369)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(8178..8266,1..994))
                     /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 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
       61 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
      121 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
      181 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
      241 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
      301 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
      361 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
      421 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
      481 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
      541 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
      601 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
      661 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
      721 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
      781 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
      841 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
      901 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
      961 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     1021 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     1081 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     1141 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     1201 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     1261 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     1321 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
     1381 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
     1441 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
     1501 agcggcctgg tgccgcgcgg cagccatatg gctagcatgt cggactcaga agtcaatcaa
     1561 gaagctaagc cagaggtcaa gccagaagtc aagcctgaga ctcacatcaa tttaaaggtg
     1621 tccgatggat cttcagagat cttcttcaag atcaaaaaga ccactccttt aagaaggctg
     1681 atggaagcgt tcgctaaaag acagggtaag gaaatggact ccttaagatt cttgtacgac
     1741 ggtattagaa tccaagctga tcagacccct gaagatttgg acatggagga taacgatatt
     1801 attgaggctc acagagaaca gattggtgga tccatggaca aagattgcga aatgaaacgt
     1861 accaccctgg atagcccgct gggcaaactg gaactgagcg gctgcgaaca gggcctgcat
     1921 gaaattaaac tgctgggtaa aggcaccagc gcggccgatg cggttgaagt tccggccccg
     1981 gccgccgtgc tgggtggtcc ggaaccgctg atgcaggcga ccgcgtggct gaacgcgtat
     2041 tttcatcagc cggaagcgat tgaagaattt ccggttccgg cgctgcatca tccggtgttt
     2101 cagcaggaga gctttacccg tcaggtgctg tggaaactgc tgaaagtggt taaatttggc
     2161 gaagtgatta gctatcagca gctggcggcc ctggcgggta atccggcggc caccgccgcc
     2221 gttaaaaccg cgctgagcgg taacccggtg ccgattctga ttccgtgcca tcgtgtggtt
     2281 agctctagcg gtgcggttgg cggttatgaa ggtggtctgg cggtgaaaga gtggctgctg
     2341 gcccatgaag gtcatcgtct gggtaaaccg ggtctgggag gtagcggtag cggtagcggt
     2401 ggaggtggaa gcagcggatc catggctctg gacgggataa ggatgccaga tggctgctac
     2461 gcggacggga cgtgggaact gagtgtccat gtgacggacc tgaaccgcga tgtcaccctg
     2521 agagtgaccg gcgaggtgca cattggaggc gtgatgctta agctggtgga gaaactcgat
     2581 gtaaaaaaag attggtctga ccatgctctc tggtgggaaa agaagagaac ttggcttctg
     2641 aagacacatt ggaccttaga taagtatggt attcaggcag atgctaagct tcagttcacc
     2701 cctcagcaca aactgctccg cctgcagctt cccaacatga agtatgtgaa ggtgaaagtg
     2761 aatttctctg atagagtctt caaagctgtt tctgacatct gtaagacttt taatatcaga
     2821 caccccgaag aactttctct cttaaagaaa cccagagatc caacaaagaa aaaaaagaag
     2881 aagctagatg accagtctga agatgaggca cttgaattag aggggcctct tatcactcct
     2941 ggatcaggaa gtatatattc aagcccagga ctgtatagta aaacaatgac ccccacttat
     3001 gatgctcatg atggaagccc cttgtcacca acttctgctt ggtttggtga cagtgctttg
     3061 tcagaaggca atcctggtat acttgctgtc agtcaaccaa tcacgtcacc agaaatcttg
     3121 gcaaaaatgt tcaagcctca agctcttctt gataaagcaa aaatcaacca aggatggctt
     3181 gattcctcaa gatctctcat ggaacaagat gtgaaggaaa atgaggcctt gctgctccga
     3241 ttcaagtatt acagcttttt tgatttgaat ccaaagtatg atgcaatcag aatcaatcag
     3301 ctttatgagc aggccaaatg ggccattctc ctggaagaga ttgaatgcac agaagaagaa
     3361 atgatgatgt ttgcagccct gcagtatcat atcaataagc tgtcaatcat gacatcagag
     3421 aatcatttga acaacagtga caaagaagtt gatgaagttg atgctgccct ttcagacctg
     3481 gagattactc tggaaggggg taaaacgtca acaattttgg gtgacattac ttccattcct
     3541 gaacttgctg actacattaa agttttcaag ccaaaaaagc tgactctgaa aggttacaaa
     3601 caatattggt gcaccttcaa agacacatcc atttcttgtt ataagagcaa agaagaatcc
     3661 agtggcacac cagctcatca gatgaacctc aggggatgtg aagttacccc agatgtaaac
     3721 atttcaggcc aaaaatttaa cattaaactc ctgattccag ttgcagaagg catgaatgaa
     3781 atctggcttc gttgtgacaa tgaaaaacag tatgcacact ggatggcagc ctgcagatta
     3841 gcctccaaag gcaagaccat ggcggacagt tcttacaact tagaagttca gaatattctt
     3901 tcctttctga agatgcagca tttaaaccca gatcctcagt taataccaga gcagatcacg
     3961 actgatataa ctcctgaatg tttggtgtct ccccgctatc taaaaaagta taagaacaag
     4021 cagataacag cgagaatctt ggaggcccat cagaatgtag ctcagatgag tctaattgaa
     4081 gccaagatga gatttattca agcttggcag tcactacctg aatttggcat cactcacttc
     4141 attgcaaggt tccaaggggg caaaaaagaa gaacttattg gaattgcata caacagactg
     4201 attcggatgg atgccagcac tggagatgca attaaaacat ggcgtttcag caacatgaaa
     4261 cagtggaatg tcaactggga aatcaaaatg gtcaccgtag agtttgcaga tgaagtacga
     4321 ttgtccttca tttgtactga agtagattgc aaagtggttc atgagttcat tggtggctac
     4381 atatttctct caacacgtgc aaaagaccaa aacgagagtt tagatgaaga gatgttctac
     4441 aaacttacca gtggttgggt gtagtaataa gaattcgagc tccgtcgaca agcttgcggc
     4501 cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga
     4561 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     4621 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac
     4681 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     4741 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     4801 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     4861 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     4921 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4981 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     5041 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     5101 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     5161 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt
     5221 aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     5281 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     5341 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     5401 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     5461 acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca
     5521 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     5581 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     5641 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     5701 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     5761 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     5821 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     5881 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     5941 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     6001 cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
     6061 tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt
     6121 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     6181 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     6241 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     6301 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     6361 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     6421 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     6481 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     6541 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     6601 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     6661 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     6721 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     6781 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     6841 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     6901 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     6961 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     7021 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     7081 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     7141 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     7201 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     7261 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     7321 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     7381 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     7441 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     7501 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     7561 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     7621 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     7681 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     7741 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     7801 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg
     7861 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     7921 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     7981 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     8041 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     8101 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     8161 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     8221 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgg
//
