LOCUS       sequence-424834-        4476 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of CENP-T[1-242/3D].
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4476)
  TITLE     Cheeseman lab plasmids Nov 2023
REFERENCE   2  (bases 1 to 4476)
  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"
COMMENT     Sequence Label: pGS275_6xHis-EGFP-CENP-T[1-242/3D (T11, T27, T...
FEATURES             Location/Qualifiers
     source          1..4476
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..46
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        27..45
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             77..99
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             119..136
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             140..172
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(176..184,185..199)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(242..244,245..247,248..958)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(242..244,245..247,248..958)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(287..308)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(548..567)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     895..916
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1004..1024
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1037..1081
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1884..1902)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1898..1945
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2042..2497
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2129..2148)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2339..2360
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2523..2627
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2628..2696,2697..3488)
                     /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     complement(2846..2865)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3659..4247
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4148..4167
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4401..4418
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aatgcaggat ctcgatcccg cgaaattaat acgactcact atagggagac cacaacggtt
       61 tccctctaga aataattttg tttaacttta agaaggagat atacatatgc ggggttctca
      121 tcatcatcat catcatggta tggctagcat gactggtgga cagcaaatgg gtcgggatct
      181 gtacgacgat gacgataagg atcgatgggg atccgagctc gagatctgca gctggtacca
      241 gatggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
      301 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
      361 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
      421 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
      481 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
      541 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
      601 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
      661 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
      721 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
      781 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
      841 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
      901 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagtc
      961 cggaggggat cggtggagct ccaccggtgg cggccgctct agagagaatc tttattttca
     1021 gggcgccgcc aaattcaaag aaaccgctgc tgctaaattc gaacgccagc acatggacag
     1081 cggaggtgga ggtactagta tggcagacca caaccctgac agcgattctg accctcgtac
     1141 actgctgcgt cgtgtgctgg atactgctga tcctcgtgat cctcgtcgtc ctcgtagcgc
     1201 tcgtgctggt gctcgccgtg ccctgctgga aacagcctct cctcgtaaac tgagtggcca
     1261 gacccgtact attgcccgtg ggcgttcaca tggagcccgc tcggtcggtc gtagtgctca
     1321 cattcaagcc tcgggacacc tggaagaaca agaccctcgt accctgctga aaaacatcct
     1381 gctgactgcc cctgaaagct ctattctgat gccggaaagc gtcgttaaac cggttcctgc
     1441 tcctcaagcc gttcaacctt ctcgtcaaga gtcctcctgt ggatctctgg agctgcaact
     1501 gcctgaactg gagcctccta caacactggc accggggctg ctggcgcctg gtcgtcgtaa
     1561 acaacgtctg cgtctgagcg tgtttcaaca aggtgttgat cagggcctga gtctgtctca
     1621 agaacctcaa ggcaacgctg atgcttctag tctgacccgt tctctgaacc tgacctttgc
     1681 tactccactg caacctcaaa gcgtccaacg tcctggactg gctcgccgcc ctcctgctcg
     1741 ccgtgccgtt gatgtgggtg cttttctgcg tgacctgcgt gatacaagcc tggcacctcc
     1801 gaatattgtg ctggaggata cacagtgaaa gcttgatccg gctgctaaca aagcccgaaa
     1861 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     1921 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat ctggcgtaat
     1981 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     2041 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2101 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2161 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     2221 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     2281 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     2341 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     2401 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     2461 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     2521 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     2581 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     2641 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     2701 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     2761 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     2821 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     2881 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     2941 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     3001 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     3061 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     3121 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     3181 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     3241 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     3301 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     3361 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     3421 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     3481 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     3541 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     3601 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3661 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     3721 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     3781 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     3841 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3901 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3961 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4021 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4081 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4141 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4201 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4261 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     4321 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     4381 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     4441 gcaaaccgcc tctccccgcg cgttggccga ttcatt
//
