LOCUS       sequence-475928-        7869 bp    DNA     circular SYN 12-JUN-2025
DEFINITION  Express mEGFP tagged mouse CENPT chimeric protein containing the rat
            CENPT histone fold domain and 4 non-synonymous substitutions.
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 Jun 12, 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: pALN148
FEATURES             Location/Qualifiers
     source          1..7869
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             973..1442
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(1572..1589)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1743..2331)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1823..1842)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2491..3282,3283..3351))
                     /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     3114..3133
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3352..3456)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             3487..3956
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    4021..4220
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             4421..4837
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     4767..4789
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     4805..4821
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      4872..4881
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4878..4880,4881..4883,4884..5594)
                     /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(4923..4944)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5184..5203)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5531..5552
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5640..5660
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             5673..5717
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(7305..7325)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7310..7639
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7490..7625
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(7649..7869,1..805)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             join(7649..7869,1..805)
                     /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 tccggaagtg cttgacattg gggaatttag cgagagcctg acctattgca tctcccgccg
       61 tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc
      121 ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg
      181 cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat
      241 tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt
      301 cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct
      361 cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt
      421 cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt
      481 ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc
      541 ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact
      601 ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga
      661 cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc
      721 ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag
      781 cactcgtccg agggcaaagg aatagatcga taaaataaaa gattttattt agtctccaga
      841 aaaagggggg aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat
      901 tttgcaaggc atggaaaaat acataactga gaatagagaa gttcagatca aggtcaggaa
      961 cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg
     1021 gctcagggcc aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa
     1081 gcagttcctg ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc
     1141 agcagtttct agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct
     1201 gtgccttatt tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc
     1261 cccgagctca ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact
     1321 gagtcgcccg ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt
     1381 ctcgctgttc cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt
     1441 cacatgcagc atgtatcaaa attaatttgg ttttttttct taagtattta cattaaatgg
     1501 ccatagttgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     1561 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     1621 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     1681 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1741 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     1801 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     1861 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     1921 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     1981 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2041 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2101 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2161 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     2221 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2281 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2341 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2401 gtcatgagat tatcaaaaag gatcttcacc tagatccttt tgcggccgca aatcaatcta
     2461 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     2521 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     2581 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     2641 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     2701 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     2761 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     2821 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     2881 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     2941 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     3001 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     3061 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     3121 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     3181 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     3241 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     3301 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     3361 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     3421 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     3481 tgcagcctga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag
     3541 ggccaagaac agatggaaca gctgaatatg ggccaaacag gatatctgtg gtaagcagtt
     3601 cctgccccgg ctcagggcca agaacagatg gtccccagat gcggtccagc cctcagcagt
     3661 ttctagagaa ccatcagatg tttccagggt gccccaagga cctgaaatga ccctgtgcct
     3721 tatttgaact aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct gctccccgag
     3781 ctcaataaaa gagcccacaa cccctcactc ggggcgccag tcctccgatt gactgagtcg
     3841 cccgggtacc cgtgtatcca ataaaccctc ttgcagttgc atccgacttg tggtctcgct
     3901 gttccttggg agggtctcct ctgagtgatt gactacccgt cagcgggggt ctttcatttg
     3961 ggggctcgtc cgggatcggg agacccctgc ccagggacca ccgacccacc accgggaggc
     4021 aagctggcca gcaacttatc tgtgtctgtc cgattgtcta gtgtctatga ctgattttat
     4081 gcgcctgcgt cggtactagt tagctaacta gctctgtatc tggcggaccc gtggtggaac
     4141 tgacgagttc tgaacacccg gccgcaaccc tgggagacgt cccagggact ttgggggccg
     4201 tttttgtggc ccgacctgag gaagggagtc gatgtggaat ccgaccccgt caggatatgt
     4261 ggttctggta ggagacgaga acctaaaaca gttcccgcct ccgtctgaat ttttgctttc
     4321 ggtttggaac cgaagccgcg cgtcttgtct gctgcagcgc tgcagcatcg ttctgtgttg
     4381 tctctgtctg actgtgtttc tgtatttgtc tgaaaattag ggccagactg ttaccactcc
     4441 cttaagtttg accttaggtc actggaaaga tgtcgagcgg atcgctcaca accagtcggt
     4501 agatgtcaag aagagacgtt gggttacctt ctgctctgca gaatggccaa cctttaacgt
     4561 cggatggccg cgagacggca cctttaaccg agacctcatc acccaggtta agatcaaggt
     4621 cttttcacct ggcccgcatg gacacccaga ccaggtcccc tacatcgtga cctgggaagc
     4681 cttggctttt gacccccctc cctgggtcaa gccctttgta caccctaagc ctccgcctcc
     4741 tcttcctcca tccgccccgt ctctccccct tgaacctcct cgttcgaccc cgcctcgatc
     4801 ctccctttat ccagccctca ctccttctct aggcgccggc cggatcccag tgtggtggta
     4861 cgctaccggt cgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     4921 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     4981 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     5041 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     5101 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     5161 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     5221 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     5281 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     5341 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     5401 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     5461 tgctgcccga caaccactac ctgagcaccc agtccaagct gagcaaagac cccaacgaga
     5521 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     5581 acgagctgta caagtccgga ggggatcggt ggagctccac cggtggcggc cgctctagag
     5641 agaatcttta ttttcagggc gccgccaaat tcaaagaaac cgctgctgct aaattcgaac
     5701 gccagcacat ggacagcgga ggtggaggtc tcgagatggc ggacctcagc ttttctgacg
     5761 gcgatcctac tgtacgcacg cttttgcggc gcgtgctgga gacggcggat tcgcgcactc
     5821 cgatgagacg ccggagcact cggatcaatg cacaaagaag acggtctcaa acgccatact
     5881 ctaacaggca gggtagccaa acaaagacaa gtgctaggaa gcaatcccat ggagctaggt
     5941 cagttggcag atcgactcgt gttcaaggtc gtgggcgcct ggaggaacag acaccccgga
     6001 ctctgctaag gaatatctta ctaactgccc cagaatcgtc cactgtgatg ccagacccag
     6061 tggtgaagcc agcacaagta ccagaagtgg cccggtcctc cagacgggag agcagtcggg
     6121 gaagcctgga gctacacctt cctgaactgg agcccccctc aaccctggct ccgggtctaa
     6181 cagcccctgg caaaaggaag cagaaactga gattatcagt atttcagcag gaagtggacc
     6241 aggggttgcc tctctcccaa gagccacgta ggagtaggag tgctgatgtt tcatccttgg
     6301 ctagctcctt caatcttacc tttgttctac ctggtcagcc agagacagtg gagagacctg
     6361 gtctggctcg aagacgtccc atccgccagc ttgtgaatgc tggtgctctt ttgcaggatc
     6421 tggaagacaa ttccttagcc tctgctcttc caggtgacag tcacagaaca cctgttgctg
     6481 ctctgccgat ggatgtaggc ttggaagaca cacagccttt ctcacagtcc ttggctgcct
     6541 tttcccttag tgggaaacat tctctgccca gcccttctag ccctggggtt gaagatgttg
     6601 aaagagttat gggcccacca agctcgggga ccaggctgca aagccggatg agcagatctg
     6661 gccctgctgc ctctccttcc cccttcctgg agccacagcc tccgcctgca gaaccgagag
     6721 aagcagtagg atccaacgag gctgcggagc ccaaggatca agaaggatct tcagggtatg
     6781 aggaaacttc tgctaggcca gcaagtggag aattatcctc cagcacacat gactcactcc
     6841 cagctgaaca gccacccccg tcacctggag ttgcagtcct atcttcagag cccctggagt
     6901 ctgtgacagc taagtgccca tccaggaccc aaactgcagg tcctaggcgc cgccaagacc
     6961 cccacaaggc tggactgagc cactatgtga agctcttcag tttccatact aagatgcccg
     7021 tggagaaaac ggctcttgag atggtagaga agtgcctaga cgagtatttc cagcgccttt
     7081 gcaatgatct ggaggtattt gctgctcatg ccggccgtaa gattgtgaag ccaaaggact
     7141 tattgctgct gatgcgacga cagggactag tcactgatca agtctcgcag catgtgcttg
     7201 tggagcggta cctgcccttg gagtaccggc aacaacttat cccctgtgca ttcagtggca
     7261 attctgtctt ccctgcacag taagaattct gcagtcgacc ctgtggaatg tgtgtcagtt
     7321 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     7381 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     7441 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     7501 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     7561 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     7621 aggcctaggc ttttgcaaaa agcttaccat gaaaaagcct gaactcaccg cgacgtctgt
     7681 cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg
     7741 cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa
     7801 tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc
     7861 gctcccgat
//
