LOCUS       sequence-409763-        4648 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses chimeric mouse CENP-O with rat CENP-O C-terminal RWD 
            domain; C-term tagged with GFP under T7 promoter - designed for in 
            vitro transcription.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4648)
  AUTHORS   Dudka D, Akins RB, Lampson MA
  TITLE     FREEDA: An automated computational pipeline guides experimental 
            testing of protein innovation.
  JOURNAL   J Cell Biol. 2023 Sep 4;222(9):e202212084. doi: 
            10.1083/jcb.202212084. Epub 2023 Jun 26.
  PUBMED    37358475
REFERENCE   2  (bases 1 to 4648)
  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"; journalName: "J Cell 
            Biol."; date: "2023-09-4"; volume: "222(9):e202212084. doi"; pages: 
            " 10.1083/jcb.202212084. Epub 2023 Jun 26"
COMMENT     Sequence Label: pGEMHE-MmCENP-O-R298-GFP
FEATURES             Location/Qualifiers
     source          1..4648
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(33..55)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     155..174
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     368..390
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     382..399
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     383..399
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     406..425
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        406..424
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     5'UTR           456..498
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             join(1439..1441,1442..2155)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1439..1441,1442..2152)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1481..1502)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1742..1761)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2089..2110
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     3'UTR           2167..2292
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     promoter        complement(2395..2413)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2396..2413)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2431..2447)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2431..2447)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2444..2466)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2455..2471
                     /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(2479..2485,2486..2503,2504..2509))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2524..2545
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2662..2679)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2833..3421)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2913..2932)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3592..4383,4384..4452))
                     /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     4215..4234
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4453..4557)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4625..4643
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
       61 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
      121 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
      181 gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
      241 cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
      301 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca
      361 gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta
      421 tagggcgaat taattcgagc tcggtaccca gcttgcttgt tctttttgca gaagctcaga
      481 ataaacgctc aactttggca gatcaattcc ccggtgctag atggagtctg caaatacttt
      541 gtgtccaggc aggaagtgca aaggaggtgt tttagctcac ctggaaagac tagaggctca
      601 aacgaacata tcgaatagaa agtctgaaga accagcagtt cgtaaaaagg aaagttctct
      661 tagaaccaag attcgagaac tgcggcaaca acgagataag ctgagggcag aagtgaagca
      721 gtggggagcc agagttaaag aacctcctgc caaagaagac ccaagcagaa ccgtgatcag
      781 tgaacaggaa gttctggaaa gggagtggag aaatgtggac gccattctgg aggcatatcg
      841 ctttacgggg ctcagcggga agttgaccag tcgtggcgtc tgcatgtgca tcagcacagc
      901 ctttgagggg aacctactag attcctactt tgtggacctc gtcatagaga aaccactccg
      961 gattcatcac cattccgtcc cagtcttcat ccctctggaa aagatagccg cagcacacct
     1021 acagactgat gttcagcgct tcctgtttag actctgggaa tacctaaatg cctacgcggg
     1081 gaggaagtac caggcagacc aacttgagag tgacttctgc gatgtcctca cgggaccttt
     1141 gcagagaagc gccctgtgca acttgctgtc attcacctat aaagtggaac acgggaccca
     1201 ggccttctcc tttagtgcca ggttgctgta tgaggacctc acagcagctc ttcccacagg
     1261 agtcactgtg acaaggccag caggtgtgga agcatcgtcc cctcattggg aagagcacag
     1321 agcatcccat gaaatgctct tccgtaccaa gaccctgcac caggcgtttg cttcattttc
     1381 caaagagaca gaaatgctgc atttgaacct ggtctccccg gtcggtggcg gaggtggcgt
     1441 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1501 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     1561 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     1621 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     1681 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     1741 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     1801 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1861 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     1921 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     1981 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     2041 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     2101 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtgaggtta
     2161 ccactaaacc agcctcaaga acacccgaat ggagtctcta agctacataa taccaactta
     2221 cactttacaa aatgttgtcc cccaaaatgt agccattcgt atctgctcct aataaaaaga
     2281 aagtttcttc acattctaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa amcccccccc
     2341 cccccctgca ggcgcgccgc cggcgattta aattaattaa tctagcttga gtattctata
     2401 gtgtcaccta aatagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     2461 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     2521 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     2581 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     2641 tattgggcgc tctttcgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     2701 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     2761 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     2821 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     2881 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     2941 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     3001 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     3061 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     3121 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     3181 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     3241 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     3301 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     3361 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     3421 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     3481 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     3541 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     3601 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     3661 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     3721 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     3781 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     3841 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     3901 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     3961 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     4021 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     4081 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     4141 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     4201 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     4261 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     4321 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     4381 gtgagcaaaa acaggaagac aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     4441 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     4501 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     4561 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     4621 taaaaatagg cgtatcacga ggcccttt
//
