LOCUS       sequence-475935-        7869 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express mEGFP tagged mouse CENPT chimeric protein containing the rat
            CENPT histone fold domain .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7869)
  AUTHORS   Dudka D, Nguyen AL, Boese KG, Marescal O, Akins RB, Black BE, 
            Cheeseman IM, Lampson MA
  TITLE     Adaptive evolution of CENP-T modulates centromere binding.
  JOURNAL   Curr Biol. 2025 Mar 10;35(5):1012-1022.e5. doi: 
            10.1016/j.cub.2025.01.017. Epub 2025 Feb 12.
  PUBMED    39947176
REFERENCE   2  (bases 1 to 7869)
  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.cub.2025.01.017"; journalName: "Curr Biol"; date: 
            "2025-03-10- 10"; volume: "35"; issue: "5"; pages: "1012-1022.e5"
COMMENT     Sequence Label: pALN132
FEATURES             Location/Qualifiers
     source          1..7869
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1062..1531
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(1661..1678)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1832..2420)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1912..1931)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2580..3371,3372..3440))
                     /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     3203..3222
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3441..3545)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             3576..4045
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    4110..4309
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             4510..4926
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     4856..4878
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     4894..4910
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      4961..4970
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4967..4969,4970..4972,4973..5683)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5012..5033)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5273..5292)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5620..5641
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5729..5749
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             5762..5806
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(7394..7414)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7399..7728
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7579..7714
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(7738..7869,1..894)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             join(7738..7869,1..894)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
ORIGIN
        1 ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat
       61 cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaatttagc
      121 gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct
      181 gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg
      241 gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac
      301 actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact
      361 gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg
      421 gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc
      481 ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat
      541 tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag
      601 cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg
      661 tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat
      721 gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc
      781 gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta
      841 ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga atagatcgat
      901 aaaataaaag attttattta gtctccagaa aaagggggga atgaaagacc ccacctgtag
      961 gtttggcaag ctagcttaag taacgccatt ttgcaaggca tggaaaaata cataactgag
     1021 aatagagaag ttcagatcaa ggtcaggaac agatggaaca gctgaatatg ggccaaacag
     1081 gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg gaacagctga
     1141 atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac
     1201 agatggtccc cagatgcggt ccagccctca gcagtttcta gagaaccatc agatgtttcc
     1261 agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca atcagttcgc
     1321 ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc cacaacccct
     1381 cactcggggc gccagtcctc cgattgactg agtcgcccgg gtacccgtgt atccaataaa
     1441 ccctcttgca gttgcatccg acttgtggtc tcgctgttcc ttgggagggt ctcctctgag
     1501 tgattgacta cccgtcagcg ggggtctttc acatgcagca tgtatcaaaa ttaatttggt
     1561 tttttttctt aagtatttac attaaatggc catagttgca ttaatgaatc ggccaacgcg
     1621 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     1681 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     1741 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     1801 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     1861 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     1921 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     1981 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     2041 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     2101 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     2161 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     2221 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     2281 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     2341 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     2401 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     2461 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     2521 agatcctttt gcggccgcaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     2581 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     2641 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     2701 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     2761 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     2821 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     2881 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     2941 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     3001 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     3061 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     3121 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     3181 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     3241 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     3301 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     3361 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     3421 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     3481 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     3541 ccgcgcacat ttccccgaaa agtgccacct gcagcctgaa tatgggccaa acaggatatc
     3601 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggaacag ctgaatatgg
     3661 gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg
     3721 tccccagatg cggtccagcc ctcagcagtt tctagagaac catcagatgt ttccagggtg
     3781 ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg
     3841 cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg
     3901 gggcgccagt cctccgattg actgagtcgc ccgggtaccc gtgtatccaa taaaccctct
     3961 tgcagttgca tccgacttgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg
     4021 actacccgtc agcgggggtc tttcatttgg gggctcgtcc gggatcggga gacccctgcc
     4081 cagggaccac cgacccacca ccgggaggca agctggccag caacttatct gtgtctgtcc
     4141 gattgtctag tgtctatgac tgattttatg cgcctgcgtc ggtactagtt agctaactag
     4201 ctctgtatct ggcggacccg tggtggaact gacgagttct gaacacccgg ccgcaaccct
     4261 gggagacgtc ccagggactt tgggggccgt ttttgtggcc cgacctgagg aagggagtcg
     4321 atgtggaatc cgaccccgtc aggatatgtg gttctggtag gagacgagaa cctaaaacag
     4381 ttcccgcctc cgtctgaatt tttgctttcg gtttggaacc gaagccgcgc gtcttgtctg
     4441 ctgcagcgct gcagcatcgt tctgtgttgt ctctgtctga ctgtgtttct gtatttgtct
     4501 gaaaattagg gccagactgt taccactccc ttaagtttga ccttaggtca ctggaaagat
     4561 gtcgagcgga tcgctcacaa ccagtcggta gatgtcaaga agagacgttg ggttaccttc
     4621 tgctctgcag aatggccaac ctttaacgtc ggatggccgc gagacggcac ctttaaccga
     4681 gacctcatca cccaggttaa gatcaaggtc ttttcacctg gcccgcatgg acacccagac
     4741 caggtcccct acatcgtgac ctgggaagcc ttggcttttg acccccctcc ctgggtcaag
     4801 ccctttgtac accctaagcc tccgcctcct cttcctccat ccgccccgtc tctccccctt
     4861 gaacctcctc gttcgacccc gcctcgatcc tccctttatc cagccctcac tccttctcta
     4921 ggcgccggcc ggatcccagt gtggtggtac gctaccggtc gccaccatgg tgagcaaggg
     4981 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     5041 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     5101 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     5161 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     5221 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     5281 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     5341 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     5401 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     5461 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     5521 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     5581 gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     5641 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtccggag gggatcggtg
     5701 gagctccacc ggtggcggcc gctctagaga gaatctttat tttcagggcg ccgccaaatt
     5761 caaagaaacc gctgctgcta aattcgaacg ccagcacatg gacagcggag gtggaggtct
     5821 cgagatggcg gacctcagct tttctgacgg cgatcctact gtacgcacgc ttttgcggcg
     5881 cgtgctggag acggcggatt cgcgcactcc gatgagacgc cggagcactc ggatcaatgc
     5941 acaaagaaga cggtctcaaa cgccatactc taacaggcag ggtagccaaa caaagacaag
     6001 tgctaggaag caatcccatg gagctaggtc agttggcaga tcgactcgtg ttcaaggtcg
     6061 tgggcgcctg gaggaacaga caccccggac tctgctaagg aatatcttac taactgcccc
     6121 agaatcgtcc actgtgatgc cagacccagt ggtgaagcca gcacaagtac cagaagtggc
     6181 ccggtcctcc agacgggaga gcagtcgggg aagcctggag ctacaccttc ctgaactgga
     6241 gcccccctca accctggctc cgggtctaac agcccctggc aaaaggaagc agaaactgag
     6301 attatcagta tttcagcagg aagtggacca ggggttgcct ctctcccaag agccacgtag
     6361 gagtaggagt gctgatgttt catccttggc tagctccttc aatcttacct ttgttctacc
     6421 tggtcagcca gagacagtgg agagacctgg tctggctcga agacgtccca tccgccagct
     6481 tgtgaatgct ggtgctcttt tgcaggatct ggaagacaat tccttagcct ctgctcttcc
     6541 aggtgacagt cacagaacac ctgttgctgc tctgccgatg gatgtaggct tggaagacac
     6601 acagcctttc tcacagtcct tggctgcctt ttcccttagt gggaaacatt ctctgcccag
     6661 cccttctcgc cctggggttg aagatgttga aagagttatg ggcccaccaa gctcggggac
     6721 caggctgcaa agccggatga gcagatctgg ccctgctgcc tctccttccc ccttcctgga
     6781 gccacagcct ccgcctgcag aaccgagaga agcagtagga tccaacgagg ctgcggagcc
     6841 caaggatcaa gaaggatctt cagggtatga ggaaacttct gctaggccag caagtggaga
     6901 attatcctcc agcacacatg actcactccc agctgaacag ccacccccgt cacctggagt
     6961 tgcagtccta tcttcagagc ccctggagtc tgtgacagct aagtgcccat ccaggaccca
     7021 aactgcaggt cctaggcgcc gccaagaccc ccacaaggct ggactgagcc actatgtgaa
     7081 gctcttcagt ttccatacta agatgcccgt ggagaaagcg gctcttgaga tggtagagaa
     7141 gtgcctagac gagtatttcc agcgcctttg caatgatctg gaggtatttg ctgctcatgc
     7201 cggccgtaag actgtgaagc caaaggactt agagctgctg atgcgacgac agggactagt
     7261 cactgatcaa gtctcgctgc atgtgcttgt ggagcggtac ctgcccttgg agtaccggca
     7321 acaacttatc ccctgtgcat tcagtggcaa ttctgtcttc cctgcacagt aagaattctg
     7381 cagtcgaccc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     7441 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     7501 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     7561 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     7621 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     7681 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gcttaccatg
     7741 aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc
     7801 gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta
     7861 ggagggcgt
//
