LOCUS       sequence-484517-        6651 bp    DNA     circular SYN 27-AUG-2025
DEFINITION  Encodes wildtype human PIPK-gamma-87 and N-terminal EBFP2 tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6651)
  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 6651)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 27, 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-PIPK-gamma-87
FEATURES             Location/Qualifiers
     source          1..6651
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      2..590
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     491..510
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        765..1068
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1069..1272
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1223..1243
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1311..1320
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1317..1319,1320..1322,1323..2033)
                     /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(1362..1383)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    4143..4264
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4180..4199)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4234..4253
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4271..4726)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4408..4429)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4620..4639
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4753..4857
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4859..5216
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4859..5050
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4882..4902)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5067..5202
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5251..6045
                     /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             5251..6045
                     /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(5305..5324)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5915..5934
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6233..6252)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6277..6324
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
       61 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
      121 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
      181 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
      241 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
      301 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
      361 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
      421 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
      481 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
      541 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
      601 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
      661 cgttatcccc tgattctgtg gataaccgta ttaccgccat gcattagtta ttaatagtaa
      721 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
      781 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
      841 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      901 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      961 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
     1021 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
     1081 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
     1141 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
     1201 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     1261 ataagcagag ctggtttagt gaaccgtcag atccgctagc gctaccggtc gccaccatgg
     1321 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     1381 acgtaaacgg ccacaagttc agcgtgaggg gcgagggcga gggcgatgcc accaacggca
     1441 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     1501 tgaccaccct gagccacggc gtgcagtgct tcgcccgcta ccccgaccac atgaagcagc
     1561 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     1621 aggacgacgg cacctacaag acccgcgccg aggtgaagtt cgagggcgac accctagtga
     1681 accgcatcga gctgaagggc gtcgacttca aggaggacgg caacatcctg gggcacaagc
     1741 tggagtacaa cttcaacagc cacaacatct atatcatggc cgtcaagcag aagaacggca
     1801 tcaaggtgaa cttcaagatc cgccacaacg tggaggacgg cagcgtgcag ctcgccgacc
     1861 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac agccactacc
     1921 tgagcaccca gtccgtgctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     1981 tggagttccg caccgccgcc gggatcactc tcggcatgga cgagctgtac aagtccggac
     2041 tcagatctcg agctcaaagc ttcgaattca tggagctgga ggtaccggac gaggcggaga
     2101 gcgctgaggc gggggccgtg ccctcggagg cggcgtgggc ggcagagagc ggggcggcgg
     2161 caggtttggc tcagaagaag gcggccccaa cagaggttct gtccatgacg gcacagccgg
     2221 gccctggcca tgggaagaag ttgggccatc gaggtgtgga cgcatccggc gaaaccacct
     2281 acaagaagac cacctcctcc accctgaagg gtgccatcca gctgggcatc ggctacaccg
     2341 tgggccacct gagctccaag cccgaacgcg acgtgctcat gcaggacttc tacgtggtgg
     2401 agagcatctt cttccccagc gaaggcagca acctcacccc cgcccaccac ttccaggact
     2461 tccgcttcaa gacctatgca cctgtcgcct tccgctactt ccgggagctc tttgggatcc
     2521 ggccagatga ttacttgtac tccctgtgca atgagccgct gatcgagctg tccaacccgg
     2581 gcgccagtgg ctccctcttc tacgtcacca gcgacgacga gttcatcatc aagaccgtca
     2641 tgcacaagga ggccgagttc ctgcagaagc tgctccctgg ctactacatg aacctcaacc
     2701 agaacccgcg gacgctgctg cccaagttct atgggctgta ctgcgtgcag tcggggggca
     2761 agaacatccg cgtcgtggtc atgaacaaca tcctgccccg cgtggtcaag atgcacctca
     2821 agttcgacct caagggctcc acctacaagc ggcgcgccag caagaaggag aaggagaaga
     2881 gcttccccac ctacaaggac ctggacttca tgcaggacat gcccgagggg ctcctgctgg
     2941 acgccgacac cttcagcgcc ctggtcaaga cgctgcagcg ggactgcctg gtcctggaaa
     3001 gtttcaagat catggactac agcctgctgc tgggcgtgca caacatcgac cagcacgagc
     3061 gcgagcggca ggcgcagggc gcccagagca cctcagatga gaagcggcct gtgggccaga
     3121 aggcgctcta ctccacggcc atggagtcca tccagggtgg cgccgcgcgc ggggaggcca
     3181 tcgaatcgga tgacacgatg ggcgggatcc ccgctgtgaa cggccgcggg gagcggctgc
     3241 tgctgcacat tggcatcatc gacatcctgc agtcctacag gttcatcaag aaactggagc
     3301 acacctggaa ggccctcgtc cacgatgggg acacggtgtc cgtccaccgc cccagcttct
     3361 atgccgagcg ctttttcaag ttcatgagca acacggtctt tcggaagaac tcctccctga
     3421 agtcctcgcc ctccaagaag gggcgcggcg gagccttgct agctgtgaaa ccgctggggc
     3481 ccaccgctgc cttctcggcc agccagatcc ctagcgagcg ggaggaggcc cagtacgacc
     3541 tgcggggggc ccgcagctac cccacgctgg aggacgaagg ccggcccgac ctcctgccct
     3601 gcacgccacc ttctttcgaa gaagccacta cagcctccat tgccacgact ctgtcatcca
     3661 catccctctc cattcctgag cggtccccct cggagacgtc ggagcagccg cggtacaggc
     3721 ggcgcacaca gtcgtctgga caggatggca ggccgcagga ggagccaccc gcggaagagg
     3781 atctgcagca gattacagtg caggtggagc ctgcgtgcag cgtggagatt gtggtcccca
     3841 aagaggagga cgcaggggtg gaggcttccc cggccggtgc ctctgctgct gttgaagtag
     3901 aaactgccag ccaggcctca gacgaggagg gcgcacctgc cagccaggcc tcggacgagg
     3961 aggacgcgcc cgccaccgac atctactttt gagtcgacgg taccgcgggc ccgggatcca
     4021 ccggatctag ataactgatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
     4081 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
     4141 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4201 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4261 cttaacgcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     4321 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     4381 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     4441 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     4501 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     4561 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     4621 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     4681 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt
     4741 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     4801 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtcct
     4861 gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     4921 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     4981 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     5041 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     5101 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     5161 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagatc
     5221 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     5281 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     5341 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     5401 accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg
     5461 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     5521 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     5581 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     5641 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     5701 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     5761 ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     5821 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     5881 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     5941 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     6001 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     6061 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     6121 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     6181 agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta actgaaacac
     6241 ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa
     6301 cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg
     6361 tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac
     6421 cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc
     6481 ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt catttttaat
     6541 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     6601 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct t
//
