LOCUS       sequence-409738-        4650 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses chimeric rat CENP-O with mouse CENP-O middle region 
            including central 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 4650)
  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 4650)
  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-RnCENP-O-203-GFP
FEATURES             Location/Qualifiers
     source          1..4650
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     33..51
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(89..111)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     211..230
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     424..446
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     438..455
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     439..455
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     462..481
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        462..480
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     5'UTR           512..554
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             join(1495..1497,1498..2211)
                     /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(1495..1497,1498..2208)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1537..1558)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1798..1817)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2145..2166
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     3'UTR           2223..2348
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     promoter        complement(2453..2471)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2454..2471)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2489..2505)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2489..2505)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2502..2524)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2513..2529
                     /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(2537..2543,2544..2561,2562..2567))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2582..2603
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2720..2737)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2891..3479)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2971..2990)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3650..4441,4442..4510))
                     /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     4273..4292
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4511..4615)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
       61 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
      121 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      181 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      241 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      301 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      361 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      421 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac tcactatagg
      481 gcgaattaat tcgagctcgg tacccagctt gcttgttctt tttgcagaag ctcagaataa
      541 acgctcaact ttggcagatc aattccccgg tgctagatgg aatctgcaaa ttctttgtgt
      601 ccaggcacag agtgtaaagg aggtgtttta gctcacctgg aaagactaga ggctcaaatg
      661 aacagatcga atagaaagtc tgaagaacca acagttcgtg aaaaggaaag ttctcttaga
      721 accaagattc gagaactgca gcaacagcga gataggctga gggtggaagt gaagcagtgg
      781 cgagccagag ttaaagaatc ttctgccaaa gaagacccaa gcagaaccgt gatcagtgaa
      841 caggaagttc tggaaaggga gtggagaaat gtggacgcca ttctggaggc atatcgcttt
      901 acggggctca gcgggaagtt gaccagtcgt ggcgtctgca tgtgcatcag cacagccttt
      961 gaggggaacc tactagattc ctactttgtg gacctcgtca tagagaaacc actccggatt
     1021 catcaccatt ccgtcccagt cttcatccct ctggaaaaga tagccgcagc acacctacag
     1081 actgatgttc agcgcttcct gtttagactc tgggaatacc taaatgccta cgcggggagg
     1141 aagtaccagg cagaccaact tgagagtgac ttctgcgatg tcctcacggg acctttgcag
     1201 agaagcgccc tgtgcaactt gctgtcattc acctataaag tggaacacgg gacccaggcc
     1261 ttctccttta gtgccaggtt gctgtatgag gacctcacag cagctcttcc cacaggagtc
     1321 actgtgacaa ggccagcagg tgtggaagca tcgtcccctc attgggaaga gcacagagca
     1381 tcccatgaaa tgctcttccg taccaagacc ctgcaccagg cgtttgcttc attttccaaa
     1441 gagacagaaa tgctgcattt gaacctggtc tccccggtcg gtggcggagg tggcgtgagc
     1501 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     1561 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     1621 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1681 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     1741 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     1801 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1861 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     1921 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     1981 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     2041 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     2101 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     2161 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagtg aggttaccac
     2221 taaaccagcc tcaagaacac ccgaatggag tctctaagct acataatacc aacttacact
     2281 ttacaaaatg ttgtccccca aaatgtagcc attcgtatct gctcctaata aaaagaaagt
     2341 ttcttcacat tctaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaamcc cccccccccc
     2401 cccctgcagg cgcgccgccg gcgatttaaa ttaattaatc tagcttgagt attctatagt
     2461 gtcacctaaa tagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     2521 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     2581 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2641 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2701 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     2761 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     2821 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     2881 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     2941 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     3001 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     3061 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     3121 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     3181 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     3241 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     3301 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     3361 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     3421 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     3481 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     3541 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     3601 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     3661 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     3721 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     3781 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     3841 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     3901 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     3961 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     4021 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     4081 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     4141 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     4201 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     4261 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     4321 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     4381 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     4441 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     4501 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     4561 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     4621 ttccccgaaa agtgccacct gacgtctaag
//
