LOCUS       sequence-348233-        6066 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mouse ILK with R255A and R349A point mutations cloned into pEGFP-N1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6066)
  AUTHORS   Martin IM, Nava MM, Wickstrom SA, Grater F
  TITLE     ATP allosterically stabilizes integrin-linked kinase for efficient 
            force generation.
  JOURNAL   Proc Natl Acad Sci U S A. 2022 Mar 15;119(11):e2106098119. doi: 
            10.1073/pnas.2106098119. Epub 2022 Mar 8.
  PUBMED    35259013
REFERENCE   2  (bases 1 to 6066)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2022-03-15"; volume: "119(11):e2106098119. 
            doi"; pages: " 10.1073/pnas.2106098119. Epub 2022 Mar 8"
COMMENT     Sequence Label: mILK-R255A/R349A-EFGP
FEATURES             Location/Qualifiers
     source          1..6066
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             74..868
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             74..868
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(128..147)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     738..757
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1056..1075)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1100..1147
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1476..2064
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1965..1984
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2239..2542
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2543..2746
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2697..2717
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      4184..4193
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4190..4192,4193..4195,4196..4909)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4190..4192,4193..4195,4196..4906)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4235..4256)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4496..4515)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4843..4864
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5032..5153
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5069..5088)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5123..5142
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5160..5615)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5297..5318)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5509..5528
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5642..5746
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(5748..6066,1..39)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5748..5939
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5771..5791)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      join(5956..6066,1..25)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg
       61 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
      121 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
      181 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
      241 cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
      301 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
      361 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
      421 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
      481 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
      541 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
      601 gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
      661 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
      721 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
      781 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
      841 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
      901 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
      961 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     1021 ctggagttct tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg
     1081 gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg
     1141 tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga
     1201 ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg
     1261 ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt
     1321 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     1381 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     1441 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     1501 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     1561 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     1621 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     1681 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     1741 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     1801 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     1861 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     1921 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     1981 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     2041 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     2101 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     2161 cgtattaccg ccatgcatta gttattaata gtaatcaatt acggggtcat tagttcatag
     2221 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     2281 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     2341 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     2401 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     2461 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     2521 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
     2581 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     2641 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     2701 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg
     2761 tcagatccgc tagcgctacc ggactcagat ctcgagctca agcttcgaat tcaccatgga
     2821 cgacattttc actcagtgcc gggagggcaa cgcggtggcg gtgcgcttgt ggctggacaa
     2881 cacagagaac gacctcaatc agggggatga tcatggcttc tcccccttgc actgggcctg
     2941 ccgagaaggc cgctctgcgg tggttgaaat gctgatcatg cgtggagcac ggattaatgt
     3001 gatgaatcgt ggggatgata cccccctgca cctggcagct agtcatggac accgtgacat
     3061 tgtacagaag ctgttgcaat acaaggctga catcaatgca gtgaatgagc acggcaatgt
     3121 gccacttcac tatgcatgtt tctggggcca agaccaggtg gcagaggacc tggtggctaa
     3181 cggggctctt gtgagcatct gtaacaagta tggagagatg cctgtggaca aagccaaggc
     3241 accccttaga gagcttctcc gagaacgggc agagaaaatg ggccagaatc tcaaccgtat
     3301 tccatacaag gacacattct ggaaggggac cactcgcaca aggccccgaa atgggaccct
     3361 gaacaaacac tccggtattg acttcaaaca gctcaacttt ctggcaaagc tcaatgagaa
     3421 tcattctgga gagctttgga aaggccgctg gcagggcaat gatattgttg tgaaggtgct
     3481 gaaggttgca gactggagta caaggaaaag cagggacttc aatgaggaat gtccccggct
     3541 caggattttc tcacacccta acgtgctccc agtgctaggt gcttgccagg ctcccccagc
     3601 cccccaccca accctcatca cacactggat gccatatgga tctctctaca atgttctaca
     3661 tgaaggcacc aatttcgttg tggaccagag ccaagctgta aagtttgctt tggacatggc
     3721 aagaggcatg gcttttcttc acacactaga gcctctcata ccccggcatg cactcaatag
     3781 ccgcagtgta atgatcgatg aagatatgac tgcccgaatc agcatggctg atgttaagtt
     3841 ttctttccag tgccctgggg ccatgtatgc gcctgcctgg gtggcccctg aagccctgca
     3901 gaagaagcct gaagacacaa acagacgctc agcagacatg tggagtttcg cggtgcttct
     3961 gtgggaactg gtgacacgag aggtgccctt tgctgacctc tctaatatgg agattgggat
     4021 gaaggtggca ctggaaggcc ttcggcctac catcccacca ggtatttccc cccatgtgtg
     4081 taagctcatg aagatttgca tgaatgaaga ccctgcaaag cgacccaagt ttgacatgat
     4141 tgtgcctatc ttggagaaga tgcaggacaa gggaccaccg gtcgccacca tggtgagcaa
     4201 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     4261 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     4321 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     4381 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     4441 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     4501 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     4561 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     4621 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     4681 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     4741 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     4801 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     4861 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa gcggccgcga
     4921 ctctagatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc
     4981 ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt
     5041 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     5101 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta
     5161 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     5221 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     5281 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     5341 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     5401 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     5461 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     5521 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     5581 acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt
     5641 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5701 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag
     5761 aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     5821 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     5881 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     5941 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     6001 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     6061 cagaag
//
