LOCUS       sequence-475929-        7869 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express mEGFP tagged mouse CENPT.
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: pALN130
FEATURES             Location/Qualifiers
     source          1..7869
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1088..1557
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(1687..1704)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1858..2446)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1938..1957)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2606..3397,3398..3466))
                     /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     3229..3248
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3467..3571)
                     /gene="bla"
                     /label=AmpR promoter
     LTR             3602..4071
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    4136..4335
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             4536..4952
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     4882..4904
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     4920..4936
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      4987..4996
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4993..4995,4996..4998,4999..5709)
                     /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(5038..5059)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5299..5318)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5646..5667
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5755..5775
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             5788..5832
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(7420..7440)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7425..7754
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7605..7740
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(7764..7869,1..920)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             join(7764..7869,1..920)
                     /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 ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg
       61 gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg
      121 aagtgcttga cattggggaa tttagcgaga gcctgaccta ttgcatctcc cgccgtgcac
      181 agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg
      241 cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat
      301 tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg
      361 atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc
      421 aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc
      481 acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg
      541 actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga
      601 ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc
      661 ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc
      721 agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa
      781 tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg
      841 tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc
      901 gtccgagggc aaaggaatag atcgataaaa taaaagattt tatttagtct ccagaaaaag
      961 gggggaatga aagaccccac ctgtaggttt ggcaagctag cttaagtaac gccattttgc
     1021 aaggcatgga aaaatacata actgagaata gagaagttca gatcaaggtc aggaacagat
     1081 ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca
     1141 gggccaagaa cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt
     1201 tcctgccccg gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag
     1261 tttctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc
     1321 ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga
     1381 gctcaataaa agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc
     1441 gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc
     1501 tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcacat
     1561 gcagcatgta tcaaaattaa tttggttttt tttcttaagt atttacatta aatggccata
     1621 gttgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     1681 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     1741 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     1801 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     1861 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     1921 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     1981 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     2041 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     2101 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     2161 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     2221 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     2281 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     2341 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     2401 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     2461 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     2521 gagattatca aaaaggatct tcacctagat ccttttgcgg ccgcaaatca atctaaagta
     2581 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     2641 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     2701 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     2761 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     2821 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     2881 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     2941 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     3001 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     3061 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     3121 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     3181 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     3241 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     3301 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     3361 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     3421 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     3481 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     3541 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgcag
     3601 cctgaatatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca
     3661 agaacagatg gaacagctga atatgggcca aacaggatat ctgtggtaag cagttcctgc
     3721 cccggctcag ggccaagaac agatggtccc cagatgcggt ccagccctca gcagtttcta
     3781 gagaaccatc agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt
     3841 gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa
     3901 taaaagagcc cacaacccct cactcggggc gccagtcctc cgattgactg agtcgcccgg
     3961 gtacccgtgt atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc
     4021 ttgggagggt ctcctctgag tgattgacta cccgtcagcg ggggtctttc atttgggggc
     4081 tcgtccggga tcgggagacc cctgcccagg gaccaccgac ccaccaccgg gaggcaagct
     4141 ggccagcaac ttatctgtgt ctgtccgatt gtctagtgtc tatgactgat tttatgcgcc
     4201 tgcgtcggta ctagttagct aactagctct gtatctggcg gacccgtggt ggaactgacg
     4261 agttctgaac acccggccgc aaccctggga gacgtcccag ggactttggg ggccgttttt
     4321 gtggcccgac ctgaggaagg gagtcgatgt ggaatccgac cccgtcagga tatgtggttc
     4381 tggtaggaga cgagaaccta aaacagttcc cgcctccgtc tgaatttttg ctttcggttt
     4441 ggaaccgaag ccgcgcgtct tgtctgctgc agcgctgcag catcgttctg tgttgtctct
     4501 gtctgactgt gtttctgtat ttgtctgaaa attagggcca gactgttacc actcccttaa
     4561 gtttgacctt aggtcactgg aaagatgtcg agcggatcgc tcacaaccag tcggtagatg
     4621 tcaagaagag acgttgggtt accttctgct ctgcagaatg gccaaccttt aacgtcggat
     4681 ggccgcgaga cggcaccttt aaccgagacc tcatcaccca ggttaagatc aaggtctttt
     4741 cacctggccc gcatggacac ccagaccagg tcccctacat cgtgacctgg gaagccttgg
     4801 cttttgaccc ccctccctgg gtcaagccct ttgtacaccc taagcctccg cctcctcttc
     4861 ctccatccgc cccgtctctc ccccttgaac ctcctcgttc gaccccgcct cgatcctccc
     4921 tttatccagc cctcactcct tctctaggcg ccggccggat cccagtgtgg tggtacgcta
     4981 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     5041 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     5101 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     5161 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     5221 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     5281 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     5341 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     5401 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     5461 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     5521 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     5581 cccgacaacc actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc
     5641 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     5701 ctgtacaagt ccggagggga tcggtggagc tccaccggtg gcggccgctc tagagagaat
     5761 ctttattttc agggcgccgc caaattcaaa gaaaccgctg ctgctaaatt cgaacgccag
     5821 cacatggaca gcggaggtgg aggtctcgag atggcggacc tcagcttttc tgacggcgat
     5881 cctactgtac gcacgctttt gcggcgcgtg ctggagacgg cggattcgcg cactccgatg
     5941 agacgccgga gcactcggat caatgcacaa agaagacggt ctcaaacgcc atactctaac
     6001 aggcagggta gccaaacaaa gacaagtgct aggaagcaat cccatggagc taggtcagtt
     6061 ggcagatcga ctcgtgttca aggtcgtggg cgcctggagg aacagacacc ccggactctg
     6121 ctaaggaata tcttactaac tgccccagaa tcgtccactg tgatgccaga cccagtggtg
     6181 aagccagcac aagtaccaga agtggcccgg tcctccagac gggagagcag tcggggaagc
     6241 ctggagctac accttcctga actggagccc ccctcaaccc tggctccggg tctaacagcc
     6301 cctggcaaaa ggaagcagaa actgagatta tcagtatttc agcaggaagt ggaccagggg
     6361 ttgcctctct cccaagagcc acgtaggagt aggagtgctg atgtttcatc cttggctagc
     6421 tccttcaatc ttacctttgt tctacctggt cagccagaga cagtggagag acctggtctg
     6481 gctcgaagac gtcccatccg ccagcttgtg aatgctggtg ctcttttgca ggatctggaa
     6541 gacaattcct tagcctctgc tcttccaggt gacagtcaca gaacacctgt tgctgctctg
     6601 ccgatggatg taggcttgga agacacacag cctttctcac agtccttggc tgccttttcc
     6661 cttagtggga aacattctct gcccagccct tctcgccctg gggttgaaga tgttgaaaga
     6721 gttatgggcc caccaagctc ggggaccagg ctgcaaagcc ggatgagcag atctggccct
     6781 gctgcctctc cttccccctt cctggagcca cagcctccgc ctgcagaacc gagagaagca
     6841 gtaggatcca acgaggctgc ggagcccaag gatcaagaag gatcttcagg gtatgaggaa
     6901 acttctgcta ggccagcaag tggagaatta tcctccagca cacatgactc actcccagct
     6961 gaacagccac ccccgtcacc tggagttgca gtcctatctt cagagcccct ggagtctgtg
     7021 acagctaagt gcccatccag gacccaaact gcaggtccta ggcgccgcca agacccccac
     7081 aaggctggac tgagccccta tgtgaagttc ttcagcttct gtactaagat gcccgtggag
     7141 aagacggctc ttgagatagt agagaagtgc ctagacaagt atttccagca cctttgcaat
     7201 gacctggagg tatttgcttc tcatgctggc cgtaagattg tgaagccaga ggacttattg
     7261 ctgctgatgc gacggcaggg actagtgact gatcaagtct cgcagcatgt gctcgtggag
     7321 cggtacctgc ccttggagta ccggcaacaa cttatccctt gtgcattcag tggcaattct
     7381 gtcttccctg cacagtaaga attctgcagt cgaccctgtg gaatgtgtgt cagttagggt
     7441 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     7501 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     7561 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     7621 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     7681 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     7741 taggcttttg caaaaagctt accatgaaaa agcctgaact caccgcgacg tctgtcgaga
     7801 agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag
     7861 aatctcgtg
//
