LOCUS       sequence-245049-        6964 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP-IPNN-MCAK in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6964)
  AUTHORS   Domnitz SB, Wagenbach M, Decarreau J, Wordeman L
  TITLE     MCAK activity at microtubule tips regulates spindle microtubule 
            length to promote robust kinetochore attachment.
  JOURNAL   J Cell Biol. 2012 Apr 16;197(2):231-7. doi: 10.1083/jcb.201108147. 
            Epub 2012 Apr 9.
  PUBMED    22492725
REFERENCE   2  (bases 1 to 6964)
  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"; doi: 
            "10.1083/jcb.201108147"; journalName: "J Cell Biol"; date: 
            "2012-04-16- 16"; volume: "197"; issue: "2"; pages: "231-7"
COMMENT     Sequence Label: pMX1418
FEATURES             Location/Qualifiers
     source          1..6964
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(613..615,616..618,619..1329)
                     /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(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3550..3567
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    3752..3873
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3789..3808)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3843..3862
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3880..4335)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4017..4038)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4229..4248
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4362..4466
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4468..4825
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4468..4659
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4491..4511)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4676..4811
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4860..5654
                     /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             4860..5654
                     /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(4914..4933)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5524..5543
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5842..5861)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5886..5933
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6262..6850
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6751..6770
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggaccggt gctagcggta ccgagctcgg atccactagt ccagtgtggt
     1381 ggaattgccc ttgccatgga gtcgcttccc gctcgattgt ttcctggtct ctccatcaag
     1441 attcaacgca gtaatggctt aattcacagt gccaacataa gcacagtgaa cgtggagaaa
     1501 tcctgtgttt cagtggaatg gatagaagga ggtaacacga aaggcaaaga gattgatttt
     1561 gatgatgtgg ctgccatcaa tccggagctt ctgcagcttc ttcccttaca cccaaaggac
     1621 aatctgccac tgcaggaaaa tgtaacagtt ccgaagcaaa aacgcagatc agtgaattcc
     1681 aaaaataacg ctccaaaaga aggtcttcga agccgttcca ctcgcatgtc cactgtccca
     1741 gaggttcgaa ttgccactca ggagaatgaa atggaggtgg agctgcctgt ggctacaaac
     1801 tctcgaaagc agttttcggt tgccactggc ctccctaggc cctcctgtcc tgcaatgaca
     1861 gaattaccgt tgtcgatggt cagcgaggag gcagaagagc aagtccatcc cacccgaagc
     1921 acatcttctg caaaccctgc tcggaggaaa tcatgtattg tgaaggaaat ggaaaaaatg
     1981 aagaacaagc gggaagagaa gagggcccag aactcggaaa taagaataaa gcgagctcag
     2041 gaatatgaca gcagctttcc aaactgggaa tttgcccgga tgattaaaga attccgggtt
     2101 actatagaat gtcacccact tactctgact gatcctacgg aagagcacag gatctgcgtg
     2161 tgtgtgagga agcgcccact gaataagcaa gaactggcca agaaagaaat cgatgtgatt
     2221 tctgttccca gcaagtgttt gctctttgtg catgaaccca aattgaaagt ggacttgaca
     2281 aagtatctgg agaatcaggc attctgcttc gattttgcct ttgatgaaac agcttcaaat
     2341 gaagtcgtct acaggttcac agcaaggcca ctggtacaga cgatttttga agggggaaaa
     2401 gcaacctgct ttgcctatgg gcagacgggc agtgggaaga ctcacacaat gggtggagac
     2461 ctgtctggta aatctcagaa tacatctaaa gggatctatg caatggcttc ccgggatgtc
     2521 ttccttctga agagtcagcc tcgctaccgg aacttaaact tggaagttta tgtgacattc
     2581 tttgagatct acaatgggaa ggtgtttgat ctgctcaaca agaaggccaa gctacgtgta
     2641 ctagaagaca gcaagcagca ggtgcaggtt gtgggacttc aggagtacct ggttaactgt
     2701 gctgatgatg tcatcaagat gctcaacatg ggcagtgcct gcaggacttc tggacagact
     2761 tttgccaact ccaattcttc ccgctcccat gcctgcttcc agattcttct tcgagccaaa
     2821 gggagattgc atggcaaatt ctccttggtg gatctggcag ggaatgaacg aggggctgac
     2881 acttctagtg ctgaccgcca gactcgcatg gagggtgcag agattaacaa gtctctccta
     2941 gcactgaagg agtgcatcag ggcgctggga cagaacaagg ctcacacccc attccgtgag
     3001 agcaagctga ctcaggtgct aagggattca ttcattgggg agaactcgag gacttgcatg
     3061 attgccatga tctcaccagg cataagctcc tgtgaatata ctttaaacac gctgagatat
     3121 gcagacagag tcaaggagct gagcccccac agcgggctca gtggagagca gccaattcaa
     3181 atggaaacgg aagagatgga agccagctct aatgggacct cgctggcagt caatttcaag
     3241 gaagaagagg agctgtcttc ccagatgtcc agctttaatg aagccatgag tcagatcagg
     3301 gagctggaag agagggccat ggaggagctc agggagatca tacagcaagg accaggctgg
     3361 cttgagctct ctgagatgac tgatcagcca gactatgact tagagacctt tgtgaacaaa
     3421 gcagaatctg ccctgactca gcagactaag cacttctcag ccctgcgaga agtcatcaag
     3481 gccctgcgtg tggccatgca actggaagaa caggctagca aacagatgaa cagcaagaaa
     3541 cggcaccaac accatcacca tcatcactaa gcggccgcat cgataagctt cgaattctgc
     3601 agtcgacggt accgcgggcc cgggatccac cggatctaga taactgatca taatcagcca
     3661 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     3721 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     3781 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     3841 ttgtggtttg tccaaactca tcaatgtatc ttaacgcgta aattgtaagc gttaatattt
     3901 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     3961 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     4021 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     4081 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     4141 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     4201 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     4261 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     4321 cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     4381 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     4441 ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg
     4501 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     4561 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     4621 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     4681 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     4741 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     4801 ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca
     4861 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     4921 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     4981 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     5041 aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     5101 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     5161 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     5221 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     5281 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     5341 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg
     5401 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     5461 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     5521 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     5581 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     5641 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     5701 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     5761 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     5821 ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc
     5881 tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg
     5941 cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca
     6001 atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg
     6061 gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact
     6121 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6181 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6241 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     6301 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     6361 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     6421 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     6481 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     6541 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     6601 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     6661 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     6721 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     6781 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     6841 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     6901 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat
     6961 gcat
//
