LOCUS       sequence-409761-        4647 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mouse 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 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-MmCENP-O-GFP
FEATURES             Location/Qualifiers
     source          1..4647
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     25..43
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(81..103)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     203..222
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     416..438
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     430..447
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     431..447
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     454..473
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        454..472
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     5'UTR           504..546
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             join(1484..1486,1487..2200)
                     /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(1484..1486,1487..2197)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1526..1547)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1787..1806)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2134..2155
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     3'UTR           2212..2337
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     promoter        complement(2442..2460)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2443..2460)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2478..2494)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2478..2494)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2491..2513)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2502..2518
                     /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(2526..2532,2533..2550,2551..2556))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2571..2592
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2709..2726)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2880..3468)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2960..2979)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3639..4430,4431..4499))
                     /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     4262..4281
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4500..4604)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt
       61 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
      121 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
      181 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg
      241 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca
      301 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
      361 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
      421 tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaatta
      481 attcgagctc ggtacccagc ttgcttgttc tttttgcaga agctcagaat aaacgctcaa
      541 ctttggcaga tcaattcccc ggtgctagat ggagtctgca aatactttgt gtccaggcag
      601 gaagtgcaaa ggaggtgttt tagctcacct ggaaagacta gaggctcaaa cgaacatatc
      661 gaatagaaag tctgaagaac cagcagttcg taaaaaggaa agttctctta gaaccaagat
      721 tcgagaactg cggcaacaac gagataagct gagggcagaa gtgaagcagt ggggagccag
      781 agttaaagaa cctcctgcca aagaagaccc aagcagaacc gtgatcagtg aacaggaagt
      841 tctggaaagg gagtggagaa atgtggacgc cattctggag gcatatcgct ttacggggct
      901 cagcgggaag ttgaccagtc gtggcgtctg catgtgcatc agcacagcct ttgaggggaa
      961 cctactagat tcctactttg tggacctcgt catagagaaa ccactccgga ttcatcacca
     1021 ttccgtccca gtcttcatcc ctctggaaaa gatagccgca gcacacctac agactgatgt
     1081 tcagcgcttc ctgtttagac tctgggaata cctaaatgcc tacgcgggga ggaagtacca
     1141 ggcagaccaa cttgagagtg acttctgcga tgtcctcacg ggacctttgc agagaaacgc
     1201 cctgtgcaac ttgctgtcgt tcacctataa agtggaacag agatgccaga ccttctcctt
     1261 cagtgccagg ttgctgtatg aggaccccac agcagctctt cccacgaatg tcactgtgac
     1321 aaggccaggt gtggaggcat catcccctcc ttgggaagag cacagagcat cccatcagat
     1381 gctcttccgt accaagcccc tgcacaaggt gtttgcttca ttttccaaag aaaccgaaaa
     1441 gctgcatttg aacctggtct ccccggtcgg tggcggaggt ggcgtgagca agggcgagga
     1501 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     1561 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     1621 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     1681 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     1741 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     1801 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     1861 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     1921 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     1981 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     2041 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     2101 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     2161 cgccgggatc actctcggca tggacgagct gtacaagtga ggttaccact aaaccagcct
     2221 caagaacacc cgaatggagt ctctaagcta cataatacca acttacactt tacaaaatgt
     2281 tgtcccccaa aatgtagcca ttcgtatctg ctcctaataa aaagaaagtt tcttcacatt
     2341 ctaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaamccc cccccccccc ccctgcaggc
     2401 gcgccgccgg cgatttaaat taattaatct agcttgagta ttctatagtg tcacctaaat
     2461 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     2521 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     2581 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     2641 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     2701 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     2761 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     2821 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2881 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     2941 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     3001 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3061 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     3121 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     3181 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3241 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     3301 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     3361 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3421 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3481 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3541 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3601 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3661 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     3721 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     3781 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     3841 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     3901 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     3961 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     4021 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     4081 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     4141 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     4201 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     4261 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     4321 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     4381 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     4441 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4501 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4561 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     4621 gtgccacctg acgtctaaga aaccatt
//
