LOCUS       sequence-409734-        4650 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses chimeric rat CENP-O with mouse CENP-O N-terminus; 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-80-GFP
FEATURES             Location/Qualifiers
     source          1..4650
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     163..185
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     177..194
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     178..194
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     201..220
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        201..219
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     5'UTR           251..293
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             join(1234..1236,1237..1950)
                     /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(1234..1236,1237..1947)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1276..1297)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1537..1556)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1884..1905
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     3'UTR           1962..2087
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     promoter        complement(2192..2210)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2193..2210)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2228..2244)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2228..2244)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2241..2263)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2252..2268
                     /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(2276..2282,2283..2300,2301..2306))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2321..2342
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2459..2476)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2630..3218)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2710..2729)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3389..4180,4181..4249))
                     /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     4012..4031
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4250..4354)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4422..4440
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4478..4500)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4600..4619
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgcacagatg cgtaaggaga aaataccgca tcaggcgcca ttcgccattc aggctgcgca
       61 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
      121 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
      181 aaacgacggc cagtgaattg taatacgact cactataggg cgaattaatt cgagctcggt
      241 acccagcttg cttgttcttt ttgcagaagc tcagaataaa cgctcaactt tggcagatca
      301 attccccggt gctagatgga gtctgcaaat actttgtgtc caggcaggaa gtgcaaagga
      361 ggtgttttag ctcacctgga aagactagag gctcaaacga acatatcgaa tagaaagtct
      421 gaagaaccag cagttcgtaa aaaggaaagt tctcttagaa ccaagattcg agaactgcgg
      481 caacaacgag ataagctgag ggcagaagtg aagcagtggg gagccagagt taaagaacct
      541 cctgccaaag aagactcaag caaaaccacg cttagtgaac aggaagttct ggaaaagcag
      601 tggagaaacc tgaaagccat tctgcaggca tatcgattta cagggctcag cgggaagttg
      661 accaaccgta gagtctgtgt gtacatcagc accgcctttg aggggaacct actagattcc
      721 tactttgtgg acctcgccat agagaaacca ctccggattc atcaccattc catcccagcc
      781 ttcatccctc tggaaaagat agccacagca tacctacaga ctgacgttca gcgcctcctg
      841 tttagtctct cggagtacct aaatgcctac gtgggaagga agtatcaggc agaccaactt
      901 gagagtgact tctgcgcttt cctcacggga cctttgcaga gaagcgccct gtgcaacttg
      961 ctgtcattca cctataaagt ggaacacggg acccaggcct tctcctttag tgccaggttg
     1021 ctgtatgagg acctcacagc agctcttccc acaggagtca ctgtgacaag gccagcaggt
     1081 gtggaagcat cgtcccctca ttgggaagag cacagagcat cccatgaaat gctcttccgt
     1141 accaagaccc tgcaccaggc gtttgcttca ttttccaaag agacagaaat gctgcatttg
     1201 aacctggtct ccccggtcgg tggcggaggt ggcgtgagca agggcgagga gctgttcacc
     1261 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     1321 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     1381 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     1441 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     1501 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     1561 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     1621 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     1681 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     1741 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     1801 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     1861 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     1921 actctcggca tggacgagct gtacaagtga ggttaccact aaaccagcct caagaacacc
     1981 cgaatggagt ctctaagcta cataatacca acttacactt tacaaaatgt tgtcccccaa
     2041 aatgtagcca ttcgtatctg ctcctaataa aaagaaagtt tcttcacatt ctaaaaaaaa
     2101 aaaaaaaaaa aaaaaaaaaa aaaaaamccc cccccccccc ccctgcaggc gcgccgccgg
     2161 cgatttaaat taattaatct agcttgagta ttctatagtg tcacctaaat agcttggcgt
     2221 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     2281 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     2341 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     2401 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     2461 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     2521 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2581 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2641 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2701 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2761 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     2821 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     2881 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     2941 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3001 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3061 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3121 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3181 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3241 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3301 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3361 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     3421 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     3481 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     3541 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     3601 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     3661 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     3721 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     3781 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     3841 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     3901 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     3961 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4021 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4081 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4141 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4201 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4261 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4321 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     4381 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     4441 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     4501 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     4561 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
     4621 tgagagtgca ccatatgcgg tgtgaaatac
//
