LOCUS       sequence-409759-        4650 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses rat CENP-O 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-GFP
FEATURES             Location/Qualifiers
     source          1..4650
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..29
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(67..89)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     189..208
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     402..424
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     416..433
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     417..433
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     440..459
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        440..458
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     5'UTR           490..532
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             join(1473..1475,1476..2189)
                     /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(1473..1475,1476..2186)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1515..1536)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1776..1795)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2123..2144
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     3'UTR           2201..2326
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     promoter        complement(2431..2449)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2432..2449)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2467..2483)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2467..2483)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2480..2502)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2491..2507
                     /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(2515..2521,2522..2539,2540..2545))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2560..2581
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2698..2715)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2869..3457)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2949..2968)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3628..4419,4420..4488))
                     /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     4251..4270
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4489..4593)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg
       61 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
      121 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct
      181 taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc
      241 gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa
      301 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
      361 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
      421 aacgacggcc agtgaattgt aatacgactc actatagggc gaattaattc gagctcggta
      481 cccagcttgc ttgttctttt tgcagaagct cagaataaac gctcaacttt ggcagatcaa
      541 ttccccggtg ctagatggaa tctgcaaatt ctttgtgtcc aggcacagag tgtaaaggag
      601 gtgttttagc tcacctggaa agactagagg ctcaaatgaa cagatcgaat agaaagtctg
      661 aagaaccaac agttcgtgaa aaggaaagtt ctcttagaac caagattcga gaactgcagc
      721 aacagcgaga taggctgagg gtggaagtga agcagtggcg agccagagtt aaagaatctt
      781 ctgccaaaga agactcaagc aaaaccacgc ttagtgaaca ggaagttctg gaaaagcagt
      841 ggagaaacct gaaagccatt ctgcaggcat atcgatttac agggctcagc gggaagttga
      901 ccaaccgtag agtctgtgtg tacatcagca ccgcctttga ggggaaccta ctagattcct
      961 actttgtgga cctcgccata gagaaaccac tccggattca tcaccattcc atcccagcct
     1021 tcatccctct ggaaaagata gccacagcat acctacagac tgacgttcag cgcctcctgt
     1081 ttagtctctc ggagtaccta aatgcctacg tgggaaggaa gtatcaggca gaccaacttg
     1141 agagtgactt ctgcgctttc ctcacgggac ctttgcagag aagcgccctg tgcaacttgc
     1201 tgtcattcac ctataaagtg gaacacggga cccaggcctt ctcctttagt gccaggttgc
     1261 tgtatgagga cctcacagca gctcttccca caggagtcac tgtgacaagg ccagcaggtg
     1321 tggaagcatc gtcccctcat tgggaagagc acagagcatc ccatgaaatg ctcttccgta
     1381 ccaagaccct gcaccaggcg tttgcttcat tttccaaaga gacagaaatg ctgcatttga
     1441 acctggtctc cccggtcggt ggcggaggtg gcgtgagcaa gggcgaggag ctgttcaccg
     1501 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     1561 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     1621 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     1681 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     1741 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     1801 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     1861 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     1921 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     1981 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     2041 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     2101 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     2161 ctctcggcat ggacgagctg tacaagtgag gttaccacta aaccagcctc aagaacaccc
     2221 gaatggagtc tctaagctac ataataccaa cttacacttt acaaaatgtt gtcccccaaa
     2281 atgtagccat tcgtatctgc tcctaataaa aagaaagttt cttcacattc taaaaaaaaa
     2341 aaaaaaaaaa aaaaaaaaaa aaaaamcccc cccccccccc cctgcaggcg cgccgccggc
     2401 gatttaaatt aattaatcta gcttgagtat tctatagtgt cacctaaata gcttggcgta
     2461 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     2521 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     2581 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     2641 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     2701 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     2761 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     2821 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     2881 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     2941 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3001 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3061 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3121 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3181 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     3241 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     3301 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     3361 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     3421 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     3481 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     3541 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     3601 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     3661 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     3721 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     3781 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     3841 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     3901 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     3961 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4021 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4081 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     4141 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     4201 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     4261 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     4321 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     4381 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     4441 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     4501 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     4561 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     4621 cgtctaagaa accattatta tcatgacatt
//
