LOCUS       sequence-123500-        7797 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Mammalian Expression of CENP-T aa 1-375.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7797)
  TITLE     Cheeseman Lab Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7797)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pKG194
FEATURES             Location/Qualifiers
     source          1..7797
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(613..615,616..618,619..1329)
                     /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(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1375..1395
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1408..1452
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2654..2673)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3715..3735
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          3897..3962
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     polyA_signal    4121..4242
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4585..4706
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(4713..5168)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4850..4871)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5062..5081
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5195..5299
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5301..5658
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5301..5492
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5324..5344)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5509..5644
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5693..6487
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             5693..6487
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5747..5766)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6357..6376
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6675..6694)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6719..6766
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7095..7683
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7584..7603
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggagggga tcggtggagc tccaccggtg gcggccgctc tagagagaat
     1381 ctttattttc agggcgccgc caaattcaaa gaaaccgctg ctgctaaatt cgaacgccag
     1441 cacatggaca gcggaggtgg aggtactagt ctcgagatgg cagaccacaa ccctgacagc
     1501 gattctacac ctcgtacact gctgcgtcgt gtgctggata ctgctgatcc tcgtactcct
     1561 cgtcgtcctc gtagcgctcg tgctggtgct cgccgtgccc tgctggaaac agcctctcct
     1621 cgtaaactga gtggccagac ccgtactatt gcccgtgggc gttcacatgg agcccgctcg
     1681 gtcggtcgta gtgctcacat tcaagcctcg ggacacctgg aagaacaaac ccctcgtacc
     1741 ctgctgaaaa acatcctgct gactgcccct gaaagctcta ttctgatgcc ggaaagcgtc
     1801 gttaaaccgg ttcctgctcc tcaagccgtt caaccttctc gtcaagagtc ctcctgtgga
     1861 tctctggagc tgcaactgcc tgaactggag cctcctacaa cactggcacc ggggctgctg
     1921 gcgcctggtc gtcgtaaaca acgtctgcgt ctgagcgtgt ttcaacaagg tgttgatcag
     1981 ggcctgagtc tgtctcaaga acctcaaggc aacgctgatg cttctagtct gacccgttct
     2041 ctgaacctga cctttgctac tccactgcaa cctcaaagcg tccaacgtcc tggactggct
     2101 cgccgccctc ctgctcgccg tgccgttgat gtgggtgctt ttctgcgtga cctgcgtgat
     2161 acaagcctgg cacctccgaa tattgtgctg gaggatacac agccgttttc acagcctatg
     2221 gttggtagcc ctaacgttta tcactcactg ccgtgtaccc ctcacactgg tgccgaagat
     2281 gctgaacaag ctgctggtcg taaaacccaa agctctggac cgggactgca aaaaaactct
     2341 ccggggaaac ctgcccaatt cctggcgggt gaagctgaag aagtcaacgc ctttgctctg
     2401 ggatttctgt ctaccagctc gggtgttagc ggagaagatg aggtggaacc tctgcatgat
     2461 ggtgtggaag aagccgagaa gaaaatggaa gaggaagggg tgtctgtctc tgaaatggaa
     2521 gcgactggtg ctcaaggacc aagtcgtgtc gaagaggcag aaggccatac cgaggttacc
     2581 gaagccgaag gaagccaagg ggaattcccg cggggaagtg gaagtggatc catggtgaaa
     2641 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc
     2701 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg
     2761 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg
     2821 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg
     2881 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc
     2941 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg
     3001 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat
     3061 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc
     3121 catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc
     3181 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat
     3241 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc
     3301 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg
     3361 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg
     3421 cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat
     3481 atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac
     3541 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca
     3601 ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     3661 gccgattcat taatgcagct ggcacgacag gtttcccgaa gcagcctgag gcctcctaag
     3721 aagaagagga aggtttgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc
     3781 ccaggcttta cactttatgc ttccgacctg cagccccttg gatctttgtg aaggaacctt
     3841 acttctgtgg tgtgacataa ttggacaaac tacctacaga gatttaaagc tctaaggtaa
     3901 atataaaatt tttaagtgta taatgtgtta aactactgat tctaattgtt tgtgtatttt
     3961 agatcacagt cccaaggctc atttcaggcc cctcagtcct cacagtctgt tcatgatcat
     4021 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     4081 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     4141 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     4201 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gggcccatcg
     4261 atggcactgg gatctgcgaa cgcagcaaga cgtagcccag cgcgtcggcc ccgagatgcg
     4321 ccgcgtgccg tgctggagat ggcggaccgg atggatatgt tctgccaagg gttggtttgc
     4381 gcattcacag ttctccgcaa gaattgattg gctccaattc ttggagtggt gaatccgtta
     4441 gcgaggtgcc gccctgcttc atccccgtgg cccgttgctc gcgtttgctg gcggtgtccc
     4501 cggaagaaat atatttgcat gtctttagtt ctatgatgac acaaaccccg cccagcgtct
     4561 tgtcattggc gaattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
     4621 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4681 ttgtccaaac tcatcaatgt atcttaacgc gtaaattgta agcgttaata ttttgttaaa
     4741 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     4801 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     4861 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     4921 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     4981 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     5041 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     5101 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     5161 gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     5221 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     5281 atattgaaaa aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta
     5341 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     5401 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     5461 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     5521 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     5581 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     5641 ggcctaggct tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc gcatgattga
     5701 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     5761 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     5821 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga
     5881 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     5941 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     6001 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     6061 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     6121 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     6181 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga
     6241 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     6301 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     6361 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     6421 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     6481 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     6541 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     6601 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccaccct
     6661 agggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg
     6721 gcaataaaaa gacagaataa aacgcacggt gttgggtcgt ttgttcataa acgcggggtt
     6781 cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc
     6841 cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca
     6901 gccaacgtcg gggcggcagg ccctgccata gcctcaggtt actcatatat actttagatt
     6961 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     7021 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     7081 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     7141 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     7201 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     7261 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     7321 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     7381 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     7441 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     7501 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     7561 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     7621 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     7681 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     7741 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc catgcat
//
