LOCUS       sequence-489630-        6998 bp    DNA     circular SYN 17-OCT-2025
DEFINITION  Encodes human kindlin2-3KA mutant and N-terminal EBFP2 tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6998)
  AUTHORS   Lee WN, Li J, Chen NP, Yu CH
  TITLE     Kindlin Regulates Mechanosensitive Activation and Adhesion Assembly 
            of Integrin beta6.
  JOURNAL   Adv Sci (Weinh). 2025 Jun 10:e01078. doi: 10.1002/advs.202501078.
  PUBMED    40492509
REFERENCE   2  (bases 1 to 6998)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 17, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Adv Sci 
            (Weinh). 2025 Jun 10:e01078. doi: 10.1002/advs.202501078."
COMMENT     Sequence Label: EBFP2-Kindlin2-3KA
FEATURES             Location/Qualifiers
     source          1..6998
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        764..1067
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1068..1271
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1222..1242
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1310..1319
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1316..1318,1319..1321,1322..2032)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(1361..1382)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    4489..4610
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4526..4545)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4580..4599
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4617..5072)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4754..4775)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4966..4985
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5099..5203
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5205..5562
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5205..5396
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5228..5248)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5413..5548
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5597..6391
                     /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             5597..6391
                     /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(5651..5670)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6261..6280
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6579..6598)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6623..6670
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat
      721 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      781 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      841 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      901 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      961 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     1021 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     1081 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     1141 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     1201 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     1261 taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggtcg ccaccatggt
     1321 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1381 cgtaaacggc cacaagttca gcgtgagggg cgagggcgag ggcgatgcca ccaacggcaa
     1441 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     1501 gaccaccctg agccacggcg tgcagtgctt cgcccgctac cccgaccaca tgaagcagca
     1561 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     1621 ggacgacggc acctacaaga cccgcgccga ggtgaagttc gagggcgaca ccctagtgaa
     1681 ccgcatcgag ctgaagggcg tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1741 ggagtacaac ttcaacagcc acaacatcta tatcatggcc gtcaagcaga agaacggcat
     1801 caaggtgaac ttcaagatcc gccacaacgt ggaggacggc agcgtgcagc tcgccgacca
     1861 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca gccactacct
     1921 gagcacccag tccgtgctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     1981 ggagttccgc accgccgccg ggatcactct cggcatggac gagctgtaca agtccggact
     2041 cagatctcga gctcaagctt cgaattctgc agtcgacggt accgcgggcc ctcgacccac
     2101 gcgtccggcg gcgcgaggct tcgctttcct ccagagcagc gacgcgggtg ccgccagcct
     2161 cgcctgttgc cgcgggagcc cggcgggagg agcgcggaag ggagccatgg ctctggacgg
     2221 gataaggatg ccagatggct gctacgcgga cgggacgtgg gaactgagtg tccatgtgac
     2281 ggacctgaac cgcgatgtca ccctgagagt gaccggcgag gtgcacattg gaggcgtgat
     2341 gcttaagctg gtggagaaac tcgatgtaaa aaaagattgg tctgaccatg ctctctggtg
     2401 ggaaaagaag agaacttggc ttctgaagac acattggacc ttagataagt atggtattca
     2461 ggcagatgct aagcttcagt tcacccctca gcacaaactg ctccgcctgc agcttcccaa
     2521 catgaagtat gtgaaggtga aagtgaattt ctctgataga gtcttcaaag ctgtttctga
     2581 catctgtaag acttttaata tcagacaccc cgaagaactt tctctcttaa agaaacccag
     2641 agatccaaca aagaaaaaaa agaagaagct agatgaccag tctgaagatg aggcacttga
     2701 attagagggg cctcttatca ctcctggatc aggaagtata tattcaagcc caggactgta
     2761 tagtaaaaca atgaccccca cttatgatgc tcatgatgga agccccttgt caccaacttc
     2821 tgcttggttt ggtgacagtg ctttgtcaga aggcaatcct ggtatacttg ctgtcagtca
     2881 accaatcacg tcaccagaaa tcttggcaaa aatgttcaag cctcaagctc ttcttgataa
     2941 agcaaaaatc aaccaaggat ggcttgattc ctcaagatct ctcatggaac aagatgtgaa
     3001 ggaaaatgag gccttgctgc tccgattcaa gtattacagc ttttttgatt tgaatccaaa
     3061 gtatgatgca atcagaatca atcagcttta tgagcaggcc aaatgggcca ttctcctgga
     3121 agagattgaa tgcacagaag aagaaatgat gatgtttgca gccctgcagt atcatatcaa
     3181 taagctgtca atcatgacat cagagaatca tttgaacaac agtgacaaag aagttgatga
     3241 agttgatgct gccctttcag acctggagat tactctggaa gggggtaaaa cgtcaacaat
     3301 tttgggtgac attacttcca ttcctgaact tgctgactac attaaagttt tcaagccagc
     3361 ggcgctgact ctgaaaggtt acgcgcaata ttggtgcacc ttcaaagaca catccatttc
     3421 ttgttataag agcaaagaag aatccagtgg cacaccagct catcagatga acctcagggg
     3481 atgtgaagtt accccagatg taaacatttc aggccaaaaa tttaacatta aactcctgat
     3541 tccagttgca gaaggcatga atgaaatctg gcttcgttgt gacaatgaaa aacagtatgc
     3601 acactggatg gcagcctgca gattagcctc caaaggcaag accatggcgg acagttctta
     3661 caacttagaa gttcagaata ttctttcctt tctgaagatg cagcatttaa acccagatcc
     3721 tcagttaata ccagagcaga tcacgactga tataactcct gaatgtttgg tgtctccccg
     3781 ctatctaaaa aagtataaga acaagcagat aacagcgaga atcttggagg cccatcagaa
     3841 tgtagctcag atgagtctaa ttgaagccaa gatgagattt attcaagctt ggcagtcact
     3901 acctgaattt ggcatcactc acttcattgc aaggttccaa gggggcaaaa aagaagaact
     3961 tattggaatt gcatacaaca gactgattcg gatggatgcc agcactggag atgcaattaa
     4021 aacatggcgt ttcagcaaca tgaaacagtg gaatgtcaac tgggaaatca aaatggtcac
     4081 cgtagagttt gcagatgaag tacgattgtc cttcatttgt actgaagtag attgcaaagt
     4141 ggttcatgaa ttcattggtg gctacatatt tctctcaaca cgtgcaaaag accaaaacga
     4201 gagtttagat gaagagatgt tctacaaact taccagtggt tgggtgtgaa taggaaaact
     4261 tgtaataaaa ctccacagcc ataacaatat ttaactttaa agctattgtt tcttatatgc
     4321 tgcttaataa agtaagcttg aactttaaaa aaaaaaaaaa agggcggccg ctctagataa
     4381 ctgatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca
     4441 cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
     4501 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4561 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta acgcgtaaat
     4621 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     4681 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     4741 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     4801 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     4861 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     4921 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     4981 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     5041 cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg
     5101 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5161 ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac
     5221 cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     5281 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     5341 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     5401 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     5461 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     5521 aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg
     5581 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     5641 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     5701 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     5761 tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     5821 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     5881 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     5941 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     6001 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     6061 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     6121 ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     6181 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     6241 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     6301 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     6361 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     6421 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     6481 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     6541 atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata
     6601 ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg
     6661 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     6721 agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     6781 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca
     6841 ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6901 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6961 tgagcgtcag accccgtaga aaagatcaaa ggatcttc
//
