LOCUS       sequence-377392-        7356 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses EGFP-tagged kinase-dead MASTL G44S with resistance to 
            MASTL siRNA (ACGCCTTATTCTAGCAAATTA).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7356)
  AUTHORS   Taskinen ME, Narva E, Conway JRW, Hinojosa LS, Lilla S, Mai A, De 
            Franceschi N, Elo LL, Grosse R, Zanivan S, Norman JC, Ivaska J
  TITLE     MASTL promotes cell contractility and motility through 
            kinase-independent signaling.
  JOURNAL   J Cell Biol. 2020 Jun 1;219(6). pii: 151688. doi: 
            10.1083/jcb.201906204.
  PUBMED    32311005
REFERENCE   2  (bases 1 to 7356)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Cell 
            Biol."; date: "2020-06-1"; volume: "219(6). pii: 151688. doi"; 
            pages: " 10.1083/jcb.201906204"
COMMENT     Sequence Label: pcDNA-EGFP MASTL G44S (siRNA resistant)
FEATURES             Location/Qualifiers
     source          1..7356
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        74..377
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        378..581
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     532..552
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      620..629
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(626..628,629..631,632..1342)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(626..628,629..631,632..1342)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(671..692)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(932..951)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1279..1300
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4157..4278
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4194..4213)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4248..4267
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4285..4740)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4422..4443)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4634..4653
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4767..4871
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4873..5230
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4873..5064
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4896..4916)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5081..5216
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5265..6059
                     /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             5265..6059
                     /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(5319..5338)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5929..5948
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6247..6266)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6291..6338
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6667..7255
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7156..7175
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taccgccatg cattagttat taatagtaat caattacggg gtcattagtt catagcccat
       61 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      121 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      181 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      241 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      301 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      361 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      421 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      481 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      541 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga
      601 tccgctagcg ctaccggtcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt
      661 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
      721 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
      781 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
      841 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
      901 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
      961 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     1021 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     1081 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     1141 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     1201 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     1261 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     1321 cggcatggac gagctgtaca agtccggact cagatctcga gctcaagctt ccatggatcc
     1381 caccgcggga agcaagaagg agcctggagg aggcgcggcg actgaggagg gcgtgaatag
     1441 gatcgcagtg ccaaaaccgc cctccattga ggaattcagc atagtgaagc ccattagccg
     1501 gagcgccttc gggaaagtgt atctggggca gaaaggcggc aaattgtatg cagtaaaggt
     1561 tgttaaaaaa gcagacatga tcaacaaaaa tatgactcat caggtccaag ctgagagaga
     1621 tgcactggca ctaagcaaaa gcccattcat tgtccatttg tattattcac tgcagtctgc
     1681 aaacaatgtc tacttggtaa tggaatatct tattggggga gatgtcaagt ctctcctaca
     1741 tatatatggt tattttgatg aagagatggc tgtgaaatat atttctgaag tagcactggc
     1801 tctagactac cttcacagac atggaatcat ccacagggac ttgaaaccgg acaatatgct
     1861 tatttctaat gagggtcata ttaaactgac ggattttggc ctttcaaaag ttactttgaa
     1921 tagagatatt aatatgatgg atatccttac aacaccatca atggcaaaac ctagacaaga
     1981 ttattcaaga accccaggac aagtgttatc gcttatcagc tcgttgggat ttaacacacc
     2041 aattgcagaa aaaaatcaag accctgcaaa catcctttca gcctgtctgt ctgaaacatc
     2101 acagctttct caaggactcg tatgccctat gtctgtagat caaaaggaca ctacccccta
     2161 cagctctaag ctgctaaaat catgtcttga aacagttgcc tccaacccag gaatgcctgt
     2221 gaagtgtcta acttctaatt tactccagtc taggaaaagg ctggccacat ccagtgccag
     2281 tagtcaatcc cacaccttca tatccagtgt ggaatcagaa tgccacagca gtcccaaatg
     2341 ggaaaaagat tgccaggaaa gtgatgaagc attgggccca acaatgatga gttggaatgc
     2401 agttgaaaag ttatgcgcaa aatctgcaaa tgccattgag acgaaaggtt tcaataaaaa
     2461 ggatctggag ttagctcttt ctcccattca taacagcagt gcccttccca ccactggacg
     2521 ctcttgtgta aaccttgcta aaaaatgctt ctctggggaa gtttcttggg aagcagtaga
     2581 actggatgta aataatataa atatggacac tgacacaagt cagttaggtt tccatcagtc
     2641 aaatcagtgg actgtggatt ctggtgggat atctgaagag caccttggga aaagaagttt
     2701 aaaaagaaat tttgagttgg ttgactccag tccttgtaaa aaaattatac agaataaaaa
     2761 aacttgtgta gagtataagc ataacgaaat gacaaattgt tatacaaatc aaaatacagg
     2821 cttaacagtt gaagtgcagg accttaagct atcagtgcac aaaagtcaac aaaatgactg
     2881 tgctaataag gagaacattg tcaattcttt tactgataaa caacaaacac cagaaaaatt
     2941 acctatacca atgatagcaa aaaaccttat gtgtgaactc gatgaagact gtgaaaagaa
     3001 tagtaagagg gactacttaa gttctagttt tctatgttct gatgatgata gagcttctaa
     3061 aaatatttct atgaactctg attcatcttt tcctggaatt tctataatgg aaagtccatt
     3121 agaaagtcag cccttagatt cagatagaag catcaaagaa tcctcttttg aagaatcaaa
     3181 tattgaagat ccacttattg taacaccaga ttgccaagaa aagacctcac caaaaggtgt
     3241 cgagaaccct gctgtacaag agagtaacca aaaaatgtta ggtcctcctt tggaggtgct
     3301 gaaaacgtta gcctctaaaa gaaatgctgt tgcttttcga agttttaaca gtcatattaa
     3361 tgcatccaat aactcagaac catccagaat gaacatgact tctttagatg caatggatat
     3421 ttcgtgtgcc tacagtggtt catatcccat ggctataacc cctactcaaa aaagaagatc
     3481 ctgtatgcca catcagcaga ccccaaatca gatcaagtcg ggaactccat accgaactcc
     3541 gaagagtgtg agaagagggg tggcccccgt tgatgatggg cgaattctag gaaccccaga
     3601 ctaccttgca cctgagctgt tactaggcag ggcccatggt cctgcggtag actggtgggc
     3661 acttggagtt tgcttgtttg aatttctaac aggaattccc cctttcaatg atgaaacacc
     3721 acaacaagta ttccagaata ttctgaaaag agatatccct tggccagaag gtgaagaaaa
     3781 gttatctgat aatgctcaaa gtgcagtaga aatactttta accattgatg atacaaagag
     3841 agctggaatg aaagagctaa aacgtcatcc tctcttcagt gatgtggact gggaaaatct
     3901 gcagcatcag actatgcctt tcatccccca gccagatgat gaaacagata cctcctattt
     3961 tgaagccagg aatactgctc agcacctgac cgtatctgga tttagtctgt agggtaccgc
     4021 gggcccggga tccaccggat ctagataact gatcataatc agccatacca catttgtaga
     4081 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     4141 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4201 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4261 actcatcaat gtatcttaac gcgtaaattg taagcgttaa tattttgtta aaattcgcgt
     4321 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     4381 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     4441 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     4501 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     4561 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     4621 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     4681 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     4741 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     4801 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     4861 aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg
     4921 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     4981 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     5041 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     5101 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     5161 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     5221 cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     5281 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     5341 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     5401 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc
     5461 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     5521 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     5581 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     5641 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     5701 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     5761 cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg
     5821 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     5881 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     5941 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     6001 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     6061 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     6121 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     6181 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag
     6241 gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa
     6301 aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca
     6361 gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc
     6421 ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt
     6481 cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa
     6541 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6601 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     6661 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     6721 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     6781 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     6841 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     6901 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     6961 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     7021 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     7081 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     7141 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     7201 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     7261 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     7321 tcctgcgtta tcccctgatt ctgtggataa ccgtat
//
