LOCUS       sequence-343362-        7710 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mouse Meikin with Separase-resistant 
            mutations.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7710)
  AUTHORS   Maier NK, Ma J, Lampson MA, Cheeseman IM
  TITLE     Separase cleaves the kinetochore protein Meikin at the meiosis I/II 
            transition.
  JOURNAL   Dev Cell. 2021 Aug 9;56(15):2192-2206.e8. doi: 
            10.1016/j.devcel.2021.06.019. Epub 2021 Jul 30.
  PUBMED    34331869
REFERENCE   2  (bases 1 to 7710)
  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: 
            "10.1016/j.devcel.2021.06.019"; journalName: "Dev Cell"; date: 
            "2021-08-9- 9"; volume: "56"; issue: "15"; pages: "2192-2206.e8"
COMMENT     Sequence Label: 3xEGFP-mMeikin (E156R, R159E)
FEATURES             Location/Qualifiers
     source          1..7710
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             join(88..90,91..93,94..804)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTALTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMALL
                     EFVTAAGITLGMDELYK"
     CDS             join(811..813,814..816,817..1527)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(811..813,814..816,817..1527)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1534..1536,1537..1539,1540..2250)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1534..1536,1537..1539,1540..2250)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2296..2316
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2329..2373
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3739..3758)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3740..3758)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    3763..3897
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3787..3806)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3841..3860
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4010..4030)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4010..4028)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4049..4065)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4049..4065)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4062..4084)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4073..4089
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4097..4103,4104..4121,4122..4127))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4142..4163
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4280..4297)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4451..5039)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4531..4550)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5209..6000,6001..6069))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5832..5851
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6070..6174)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6200..6655)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6337..6358)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6549..6568
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6726..7710
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atccaccatg gtgagcaagg gcgaggagct gttcaccggg
      121 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
      181 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
      241 ggcaagctgc ccgtgccctg gcccaccctc gtgaccgccc tgacctacgg cgtgcagtgc
      301 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
      361 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
      421 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
      481 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
      541 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
      601 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
      661 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
      721 cccaacgaga agcgcgatca catggccctg ctggagttcg tgaccgccgc cgggatcact
      781 ctcggcatgg acgagctgta caagacgcgt atggtgagca agggcgagga gctgttcacc
      841 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
      901 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
      961 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     1021 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     1081 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     1141 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     1201 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     1261 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     1321 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     1381 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     1441 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     1501 actctcggca tggacgagct gtacaagccc gggatggtga gcaagggcga ggagctgttc
     1561 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     1621 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     1681 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     1741 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     1801 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     1861 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     1921 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     1981 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     2041 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     2101 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     2161 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     2221 atcactctcg gcatggacga gctgtacaag tccggagggg atcggtggag ctccaccggt
     2281 ggcggccgct ctagagagaa tctttatttt cagggcgccg ccaaattcaa agaaaccgct
     2341 gctgctaaat tcgaacgcca gcacatggac agcggaggtg gaggtactag tctcgagatg
     2401 gataagatct ggcacatggg acccaggggc gactacacca gaaaaaaaag ggccggcgag
     2461 aggctgaacc tgacccctaa acccgacctg gccctgcctg gaagaaccga agccctgccc
     2521 ggcctcaagg gaaagggcaa ggagcaaggc ctgaggaaga ttacagagaa gaaggagctc
     2581 agcaggctga ccggcagcag ctcccagaga ccttccctgc tcagcgtgac cggaggcgag
     2641 cacctgcagg agaacagccc cggacaggag acccccgagg aaaagacccc cccctgcgag
     2701 accgtcaccg ataccttcga gatggactcc ctgctgtcct ccaccgagct ggtgagcgga
     2761 cccgctgagc aggatgactt ctcctcctgc ctgcccagct gtagcaacgc cgagctgcat
     2821 acagaatcca cagatgagag gggcagcagc ttccccagcc ccaggctgtt cgagggcagc
     2881 gactgcctgg actgggagca tcccaagtta gaagactaca tgttctacaa aaattctacc
     2941 ctgctcgata cctccaaagc cgtggtcatc gagaaggccc cccaattcgc caatctgagc
     3001 gccgtcctct ccagctccag caagaactac gagaagagac acagaaagat tggcatgaca
     3061 ctggccgccc agcacctgag ccctgagcct aagtacgcta gcaacctggc ctccgtcgtg
     3121 gataacgccg ccagcgaagt ggtcttcgcc gagaagacag gcccccccac caccgagaag
     3181 acccagaaaa agcccgagaa tgagtccgag gacagcggcc ctctggtgca gaccaagctc
     3241 tccagcggcc atcccgacaa caaggccctg tgtagccctc tgtcctccgc cctggagtcc
     3301 accgctgtga gatacaccct gctgccccag cccctcgaac ctgtgctgaa gaagggctgc
     3361 atcctgcccg acaagcagag caaggctctg ctgacatcca caccctcctc cgatatcgcc
     3421 gagttcgtga tcgacctctc ccctgtccag aacgtgagct ttgaggagct gttccccaac
     3481 gtcagcaact atgtgaacag cagcgaagtc gtgcccgtga gcagcctcca agagagcagc
     3541 tccaatgagt tcagccccaa caccagcgaa atctgctgca tcatcaggag cagccccggc
     3601 acaaggcaga tgaggagaaa ggaccccgct gtgaagaaca gatgctcccc ccctaaggac
     3661 gtgcctctgg atattatcat gaagaccaac ggaagaacat aagaattcct taaggctagc
     3721 cctaggacgc gtcccgggcc ctatagtgag tcgtattacg tagatccaga catgataaga
     3781 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     3841 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     3901 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa
     3961 ttcgcggccg cggcgccaat gcattgggcc cggtacccag cttttgttcc ctttagtgag
     4021 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     4081 cgctcacaat tccacacaac atacgagccg ggagcataaa gtgtaaagcc tggggtgcct
     4141 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4201 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4261 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4321 gagcggtatc agctcactca aagacggtaa tacggttatc cacagaatca ggggataacg
     4381 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4441 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4501 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4561 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4621 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4681 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4741 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4801 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4861 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     4921 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4981 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5041 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5101 ggattttggt catgagatta tcaaaaggat cttcacctag atccttttaa attaaaaatg
     5161 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5221 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5281 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5341 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5401 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5461 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5521 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5581 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5641 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5701 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5761 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     5821 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     5881 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     5941 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6001 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6061 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     6121 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6181 tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     6241 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     6301 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     6361 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     6421 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     6481 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     6541 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     6601 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat
     6661 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     6721 cgccagtcga ccgccaattc aatatggcgt atatggactc atgccaattc aatatggtgg
     6781 atctggacct gtgccaattc aatatggcgt atatggactc gtgccaattc aatatggtgg
     6841 atctggaccc cagccaattc aatatggcgg acttggcacc atgccaattc aatatggcgg
     6901 acctggcact gtgccaactg gggaggggtc tacttggcac ggtgccaagt ttgaggaggg
     6961 gtcttggccc tgtgccaagt ccgccatatt gaattggcat ggtgccaata atggcggcca
     7021 tattggctat atgccaggat caatatatag gcaatatcca atatggccct atgccaatat
     7081 ggctattggc caggttcaat actatgtatt ggccctatgc catatagtat tccatatatg
     7141 ggttttccta ttgacgtaga tagcccctcc caatgggcgg tcccatatac catatatggg
     7201 gcttcctaat accgcccata gccactcccc cattgacgtc aatggtctct atatatggtc
     7261 tttcctattg acgtcatatg ggcggtccta ttgacgtata tggcgcctcc cccattgacg
     7321 tcaattacgg taaatggccc gcctggctca atgcccattg acgtcaatag gaccacccac
     7381 cattgacgtc aatgggatgg ctcattgccc attcatatcc gttctcacgc cccctattga
     7441 cgtcaatgac ggtaaatggc ccacttggca gtacatcaat atctattaat agtaacttgg
     7501 caagtacatt actattggaa gtacgccagg gtacattggc agtactccca ttgacgtcaa
     7561 tggcggtaaa tggcccgcga tggctgccaa gtacatcccc attgacgtca atggggaggg
     7621 gcaatgacgc aaatgggcgt tccattgacg taaatgggcg gtaggcgtgc ctaatgggag
     7681 gtctatataa gcaatgctcg tttagggaac
//
