LOCUS       sequence-409741-        4647 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses chimeric rat CENP-O with mouse 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 4647)
  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 4647)
  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-298-GFP
FEATURES             Location/Qualifiers
     source          1..4647
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(11..33)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     133..152
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     346..368
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     360..377
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     361..377
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     384..403
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        384..402
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     5'UTR           434..476
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             join(1414..1416,1417..2130)
                     /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(1414..1416,1417..2127)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1456..1477)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1717..1736)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2064..2085
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     3'UTR           2142..2267
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     promoter        complement(2372..2390)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2373..2390)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2408..2424)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2408..2424)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2421..2443)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2432..2448
                     /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(2456..2462,2463..2480,2481..2486))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2501..2522
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2639..2656)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2810..3398)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2890..2909)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3569..4360,4361..4429))
                     /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     4192..4211
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4430..4534)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4602..4620
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
       61 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
      121 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa
      181 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc
      241 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
      301 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
      361 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaatta attcgagctc
      421 ggtacccagc ttgcttgttc tttttgcaga agctcagaat aaacgctcaa ctttggcaga
      481 tcaattcccc ggtgctagat ggaatctgca aattctttgt gtccaggcac agagtgtaaa
      541 ggaggtgttt tagctcacct ggaaagacta gaggctcaaa tgaacagatc gaatagaaag
      601 tctgaagaac caacagttcg tgaaaaggaa agttctctta gaaccaagat tcgagaactg
      661 cagcaacagc gagataggct gagggtggaa gtgaagcagt ggcgagccag agttaaagaa
      721 tcttctgcca aagaagactc aagcaaaacc acgcttagtg aacaggaagt tctggaaaag
      781 cagtggagaa acctgaaagc cattctgcag gcatatcgat ttacagggct cagcgggaag
      841 ttgaccaacc gtagagtctg tgtgtacatc agcaccgcct ttgaggggaa cctactagat
      901 tcctactttg tggacctcgc catagagaaa ccactccgga ttcatcacca ttccatccca
      961 gccttcatcc ctctggaaaa gatagccaca gcatacctac agactgacgt tcagcgcctc
     1021 ctgtttagtc tctcggagta cctaaatgcc tacgtgggaa ggaagtatca ggcagaccaa
     1081 cttgagagtg acttctgcgc tttcctcacg ggacctttgc agagaaacgc cctgtgcaac
     1141 ttgctgtcgt tcacctataa agtggaacag agatgccaga ccttctcctt cagtgccagg
     1201 ttgctgtatg aggaccccac agcagctctt cccacgaatg tcactgtgac aaggccaggt
     1261 gtggaggcat catcccctcc ttgggaagag cacagagcat cccatcagat gctcttccgt
     1321 accaagcccc tgcacaaggt gtttgcttca ttttccaaag aaaccgaaaa gctgcatttg
     1381 aacctggtct ccccggtcgg tggcggaggt ggcgtgagca agggcgagga gctgttcacc
     1441 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     1501 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     1561 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     1621 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     1681 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     1741 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     1801 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     1861 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     1921 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     1981 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     2041 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     2101 actctcggca tggacgagct gtacaagtga ggttaccact aaaccagcct caagaacacc
     2161 cgaatggagt ctctaagcta cataatacca acttacactt tacaaaatgt tgtcccccaa
     2221 aatgtagcca ttcgtatctg ctcctaataa aaagaaagtt tcttcacatt ctaaaaaaaa
     2281 aaaaaaaaaa aaaaaaaaaa aaaaaamccc cccccccccc ccntgcaggc gcgccgccgg
     2341 cgatttaaat taattaatct agcttgagta ttctatagtg tcacctaaat agcttggcgt
     2401 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     2461 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     2521 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     2581 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     2641 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     2701 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2761 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2821 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2881 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2941 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3001 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3061 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3121 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3181 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3241 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3301 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3361 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3421 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3481 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3541 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     3601 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     3661 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     3721 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     3781 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     3841 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     3901 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     3961 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4021 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4081 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4141 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4201 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4261 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4321 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4381 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4441 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4501 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     4561 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     4621 cctttcgtct cgcgcgtttc ggtgatg
//
