LOCUS       sequence-450068-        4845 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry vector with a phospho-mimetic mutant (YYEE) of the 
            Illusia FRET reporter for integrin phosphorylation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4845)
  AUTHORS   Conway JRW, Joshi O, Kaivola J, Follain G, Gounis M, Kuhl D, Ivaska 
            J
  TITLE     Dynamic regulation of integrin beta1 phosphorylation supports 
            invasion of breast cancer cells.
  JOURNAL   Nat Cell Biol. 2025 May 26. doi: 10.1038/s41556-025-01663-4.
  PUBMED    40419795
REFERENCE   2  (bases 1 to 4845)
  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: "Nat Cell 
            Biol. 2025 May 26. doi: 10.1038/s41556-025-01663-4."
COMMENT     Sequence Label: pENTR221-Illusia(YYEE)
FEATURES             Location/Qualifiers
     source          1..4845
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     49..66
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(225..252)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(344..430)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     479..501
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     493..510
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     494..510
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    526..625
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      635..644
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(701..703,704..706,707..1414)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLLCTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLL
                     EFLTAAGITEGMNELY"
     CDS             1754..2101
                     /codon_start=1
                     /product="long flexible linker consisting of SAGG repeats 
                     (Komatsu et al., 2011)"
                     /label=EV linker
                     /translation="SAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGS
                     AGGSAGGSGSAGGSAGGSTSAGGSAGGSAGGSAGGSAGGSGSAGGSAGGSTSAGGSAGG
                     SAGGSAGGSAGG"
     CDS             join(2198..2200,2201..2203,2204..2917)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     protein_bind    complement(2927..3026)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3043..3062)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3044..3062)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3067..3083)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3067..3083)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3185..3204)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3196..4005
                     /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="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3263..3282)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4152..4740
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4641..4660
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
       61 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
      121 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
      181 attaatacgc gtaccgctag ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca
      241 ggatggcctt ctgcttagtt tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc
      301 ctccgggccg ttgcttcaca acgttcaaat ccgctcccgg cggatttgtc ctactcagga
      361 gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtcttccgac tgagcctttc
      421 gttttatttg atgcctggca gttccctact ctcgcgttaa cgctagcatg gatgttttcc
      481 cagtcacgac gttgtaaaac gacggccagt cttaagctcg ggccccaaat aatgatttta
      541 ttttgactga tagtgacctg ttcgttgcaa caaattgatg agcaatgctt ttttataatg
      601 ccaactttgt acaaaaaagc aggctagcgt cgacgccacc atgggctgca tcaagagcaa
      661 gcgcaaggac aacctgaacg acgacggcgt ggactctaga atggtgagca agggcgagga
      721 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
      781 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagct
      841 tctatgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgggcta
      901 cggcctgcag tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc
      961 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     1021 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     1081 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     1141 cagccacaac gtctatatca ccgccgacaa gcagaagaac ggcatcaagg ccaacttcaa
     1201 gatccgccac aacatcgagg acggcggcgt gcagctcgcc gaccactacc agcagaacac
     1261 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagct accagtccgc
     1321 cctgttcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcctgaccgc
     1381 cgccgggatc actgagggca tgaacgagct gtacctcgag gaaggttccc aattctgggt
     1441 aacggtgcag aggactgagg ccgccgagcg ctgtggcctg catggctcct acgtgctgag
     1501 ggtggaggct gaaaggctga ctctcctgac cgtgggggcc cagagtcaga tactggagcc
     1561 actcctgtcc tggccctaca ctctgttgcg tcgctatggc cgggacaagg tcatgttctc
     1621 tttcgaggcc ggccgccgct gcccctcagg ccctggaacc ttcaccttcc agacggcaca
     1681 gggaaatgac atcttccagg cagttgagac tgccatccac cggcagaagg cccagggaaa
     1741 ggccggaggt accagtgctg gtggtagtgc tggtggtagt gctggtggta gtgctggtgg
     1801 tagtgctggt ggttccggca gtgctggtgg tagtgctggt ggtagtacca gtgctggtgg
     1861 tagtgctggt ggtagtgctg gtggtagtgc tggtggtagt gctggtggtt ccggcagtgc
     1921 tggtggtagt gctggtggta gtaccagtgc tggtggtagt gctggtggta gtgctggtgg
     1981 tagtgctggt ggtagtgctg gtggttccgg cagtgctggt ggtagtgctg gtggtagtac
     2041 cagtgctggt ggtagtgctg gtggtagtgc tggtggtagt gctggtggta gtgctggtgg
     2101 ttccggaaaa ggagaggaaa agccgaatgt cgtgactaca gtagccagta aggaaattcc
     2161 taatgaaggt acggactgga aagccaatgg cggccgcatg gtgagcaagg gcgaggagct
     2221 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     2281 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     2341 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgtcctgggg
     2401 cgtgcagtgc ttcgcccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     2461 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     2521 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     2581 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actactttag
     2641 cgacaacgtc tatatcaccg ccgacaagca gaagaacggc atcaaggcca acttcaagat
     2701 ccgccacaac atcgaggacg gcggcgtgca gctcgccgac cactaccagc agaacacccc
     2761 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccaagct
     2821 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     2881 cgggatcact ctcggcatgg acgagctgta caagtaatga gaattcaccc agctttcttg
     2941 tacaaagttg gcattataag aaagcattgc ttatcaattt gttgcaacga acaggtcact
     3001 atcagtcaaa ataaaatcat tatttgccat ccagctgata tcccctatag tgagtcgtat
     3061 tacatggtca tagctgtttc ctggcagctc tggcccgtgt ctcaaaatct ctgatgttac
     3121 attgcacaag ataaaaatat atcatcatga acaataaaac tgtctgctta cataaacagt
     3181 aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg attaaattcc
     3241 aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt
     3301 gcgacaatct atcgcttgta tgggaagccc gatgcgccag agttgtttct gaaacatggc
     3361 aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa
     3421 tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc
     3481 accactgcga tccccggaaa aacagcattc caggtattag aagaatatcc tgattcaggt
     3541 gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt
     3601 aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat
     3661 aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa
     3721 gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt
     3781 gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt
     3841 ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt
     3901 gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat
     3961 atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga attggttaat
     4021 tggttgtaac actggcagag cattacgctg acttgacggg acggcgcaag ctcatgacca
     4081 aaatccctta acgtgagtta cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc
     4141 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4201 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4261 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     4321 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4381 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4441 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4501 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4561 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4621 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4681 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4741 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4801 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttacc
//
