LOCUS       sequence-426376-        5163 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of CENP-T[1-242/3D]-I3-01.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5163)
  TITLE     Cheeseman lab plasmids Nov 2023
REFERENCE   2  (bases 1 to 5163)
  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: pGS58_6xHis-EGFP-CENP-T[1-242/3D (T11, T27, T8...
FEATURES             Location/Qualifiers
     source          1..5163
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     23..42
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        23..41
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             73..95
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             115..132
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             136..168
                     /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(172..180,181..195)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(238..240,241..243,244..954)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(238..240,241..243,244..954)
                     /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(283..304)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(544..563)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     891..912
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1000..1020
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1033..1077
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2567..2585)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2581..2628
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2725..3180
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2812..2831)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3022..3043
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3206..3310
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3311..3379,3380..4171)
                     /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(3529..3548)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4342..4930
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4831..4850
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5084..5101
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 caggatctcg atcccgcgaa attaatacga ctcactatag ggagaccaca acggtttccc
       61 tctagaaata attttgttta actttaagaa ggagatatac atatgcgggg ttctcatcat
      121 catcatcatc atggtatggc tagcatgact ggtggacagc aaatgggtcg ggatctgtac
      181 gacgatgacg ataaggatcg atggggatcc gagctcgaga tctgcagctg gtaccagatg
      241 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
      301 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
      361 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
      421 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
      481 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
      541 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
      601 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
      661 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
      721 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
      781 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
      841 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
      901 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtccgga
      961 ggggatcggt ggagctccac cggtggcggc cgctctagag agaatcttta ttttcagggc
     1021 gccgccaaat tcaaagaaac cgctgctgct aaattcgaac gccagcacat ggacagcgga
     1081 ggtggaggta ctagtctcga gatggcagac cacaaccctg acagcgattc tgaccctcgt
     1141 acactgctgc gtcgtgtgct ggatactgct gatcctcgtg atcctcgtcg tcctcgtagc
     1201 gctcgtgctg gtgctcgccg tgccctgctg gaaacagcct ctcctcgtaa actgagtggc
     1261 cagacccgta ctattgcccg tgggcgttca catggagccc gctcggtcgg tcgtagtgct
     1321 cacattcaag cctcgggaca cctggaagaa caagaccctc gtaccctgct gaaaaacatc
     1381 ctgctgactg cccctgaaag ctctattctg atgccggaaa gcgtcgttaa accggttcct
     1441 gctcctcaag ccgttcaacc ttctcgtcaa gagtcctcct gtggatctct ggagctgcaa
     1501 ctgcctgaac tggagcctcc tacaacactg gcaccggggc tgctggcgcc tggtcgtcgt
     1561 aaacaacgtc tgcgtctgag cgtgtttcaa caaggtgttg atcagggcct gagtctgtct
     1621 caagaacctc aaggcaacgc tgatgcttct agtctgaccc gttctctgaa cctgaccttt
     1681 gctactccac tgcaacctca aagcgtccaa cgtcctggac tggctcgccg ccctcctgct
     1741 cgccgtgccg ttgatgtggg tgcttttctg cgtgacctgc gtgatacaag cctggcacct
     1801 ccgaatattg tgctggagga tacacaggaa ttcccgcggg gaagtggaag tggatccgga
     1861 ggcagcggtg gctccggggg ctctggaggc agcatgaaga tggaggagct gttcaagaag
     1921 cacaagatcg tggccgtgct gagagccaac agcgtggagg aggccaagaa gaaggccctg
     1981 gccgtgttcc tgggcggcgt gcacctgatc gagatcacct tcaccgtgcc cgacgccgac
     2041 accgtgatca aggagctgag cttcctgaag gagatgggcg ccatcatcgg cgccggcacc
     2101 gtgaccagcg tggagcagtg cagaaaggcc gtggagagcg gcgccgagtt catcgtgagc
     2161 ccccacctgg acgaggagat cagccagttc tgcaaggaga agggcgtgtt ctacatgccc
     2221 ggcgtgatga cccccaccga gctggtgaag gccatgaagc tgggccacac catcctgaag
     2281 ctgttccccg gcgaggtggt gggcccccag ttcgtgaagg ccatgaaggg ccccttcccc
     2341 aacgtgaagt tcgtgcccac cggcggcgtg aacctggaca acgtgtgcga gtggttcaag
     2401 gccggcgtgc tggccgtggg cgtgggcagc gccctggtga agggcacccc cgtggaggtg
     2461 gccgagaagg ccaaggcctt cgtggagaag atcagaggct gcaccgagtg aaagcttgat
     2521 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     2581 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     2641 actatatccg gatctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     2701 tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2761 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2821 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2881 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2941 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3001 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     3061 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3121 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     3181 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3241 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3301 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3361 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3421 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     3481 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     3541 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3601 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3661 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3721 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3781 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3841 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3901 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3961 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     4021 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     4081 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     4141 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     4201 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4261 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4321 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4381 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4441 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     4501 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4561 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4621 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4681 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4741 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4801 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4861 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4921 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4981 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     5041 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     5101 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     5161 atg
//
