LOCUS       sequence-134723-        6434 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression of EGFP-TEV-S-tag CENP-A for protein 
            localization and affinity purification (LAP).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6434)
  TITLE     Cheeseman Lab Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6434)
  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            "Unpublished"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: pKG96
FEATURES             Location/Qualifiers
     source          1..6434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             8..477
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    542..741
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             934..1350
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1318..1334
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      1385..1394
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1391..1393,1394..1396,1397..2107)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1391..1393,1394..1396,1397..2107)
                     /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(1436..1457)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1697..1716)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2044..2065
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2153..2173
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2186..2230
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2850..2870)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2855..3184
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3035..3170
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3199..3246
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     LTR             3715..4308
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(4509..4526)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4680..5268)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4760..4779)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5446..6237,6238..6306))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6069..6088
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6307..6411)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgcagcctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca
       61 gggccaagaa cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt
      121 tcctgccccg gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag
      181 tttctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc
      241 ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga
      301 gctcaataaa agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc
      361 gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc
      421 tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcattt
      481 gggggctcgt ccgggatcgg gagacccctg cccagggacc accgacccac caccgggagg
      541 taagctggcc agcaacttat ctgtgtctgt ccgattgtct agtgtctatg actgatttta
      601 tgcgcctgcg tcggtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa
      661 ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc
      721 gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg
      781 tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt
      841 cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagca tcgttctgtg ttgtctctgt
      901 ctgactgtgt ttctgtattt gtctgaaaat tagggccaga ctgttaccac tcccttaagt
      961 ttgaccttag atcactggaa agatgtcgag cggctcgctc acaaccagtc ggtagatgtc
     1021 aagaagagac gttgggttac cttctgctct gcagaatggc caacctttaa cgtcggatgg
     1081 ccgcgagacg gcacctttaa ccgagacctc atcacccagg ttaagatcaa ggtcttttca
     1141 cctggcccgc atggacaccc agaccaggtc ccctacatcg tgacctggga agccttggct
     1201 tttgaccccc ctccctgggt caagcccttt gtacacccta agcctccgcc tcctcttctt
     1261 ccatccgcgc cgtctctccc ccttgaacct cctctttcga ccccgcctca atcctccctt
     1321 tatccagccc tcactccttc tctaggcgcc ggccggatcc cagtgtggtg gtacgctacc
     1381 ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     1441 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     1501 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     1561 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     1621 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     1681 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     1741 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     1801 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     1861 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     1921 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     1981 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     2041 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     2101 gtacaagtcc ggaggggatc ggtggagctc caccggtggc ggccgctcta gagagaatct
     2161 ttattttcag ggcgccgcca aattcaaaga aaccgctgct gctaaattcg aacgccagca
     2221 catggacagc ggaggtggag gtactagtct cgagggcccg cgccgccgga gccgaaagcc
     2281 cgaggtcccg aggaggcgca gcccgagccc gaccccgacc cccggcccct cccggcgggg
     2341 cccctcctta ggcgcttcct cccatcaaca cagtcggcgg agacaaggtt ggctaaagga
     2401 gatccgaaag cttcagaaga gcacacacct cttgataagg aagctgccct tcagccgcct
     2461 ggcaagagaa atatatgtta aattcactcg tggtgtggac ttcaattggc aagcccaggc
     2521 cctattggcc ctacaagagg cagcagaagc atttctagtt catctctttg aggacgccta
     2581 tctcctcacc ttacatgcag gccgagttac tttcttccca aaggatgtgc aactggcccg
     2641 gaggatccgg ggccttgagg agggactcgg ctgagaattc tgcagtcgac ggtaccgcgg
     2701 gcccgggatc caccggatct agataactga tcataatcag ccataccaca tttgtagagg
     2761 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg
     2821 caattcgcca gcacagtggt cgaccctgtg gaatgtgtgt cagttagggt gtggaaagtc
     2881 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     2941 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3001 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     3061 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     3121 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     3181 caaaaagctt gagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa
     3241 tacgacaagg tgaggaacta aaccatggcc aagcctttgt ctcaagaaga atccaccctc
     3301 attgaaagag caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc
     3361 agcgcagctc tctctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact
     3421 gggggacctt gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac
     3481 ctgacttgta tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg
     3541 tgccgacagg tgcttctcga tctgcatcct gggatcaaag ccatagtgag gacagtgatg
     3601 gacagccgac ggcagttggg attcgtgaat tgctgccctc tggttatgtg tgggagggct
     3661 aagcacttca tcgataaaat aaaagatttt atttagtctc cagaaaaagg ggggaatgaa
     3721 agaccccacc tgtaggtttg gcaagctagc ttaagtaacg ccattttgca aggcatggaa
     3781 aaatacataa ctgagaatag agaagttcag atcaaggtca ggaacagatg gaacagctga
     3841 atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac
     3901 agatggaaca gctgaatatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg
     3961 ctcagggcca agaacagatg gtccccagat gcggtccagc cctcagcagt ttctagagaa
     4021 ccatcagatg tttccagggt gccccaagga cctgaaatga ccctgtgcct tatttgaact
     4081 aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct gctccccgag ctcaataaaa
     4141 gagcccacaa cccctcactc ggggcgccag tcctccgatt gactgagtcg cccgggtacc
     4201 cgtgtatcca ataaaccctc ttgcagttgc atccgacttg tggtctcgct gttccttggg
     4261 agggtctcct ctgagtgatt gactacccgt cagcgggggt ctttcatttc cgacttgtgg
     4321 tctcgctgcc ttgggagggt ctcctctgag tgattgacta cccgtcagcg ggggtcttca
     4381 catgcagcat gtatcaaaat taatttggtt ttttttctta agtatttaca ttaaatggcc
     4441 atagttgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttggcgctct
     4501 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4561 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4621 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4681 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4741 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4801 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4861 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4921 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4981 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5041 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5101 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5161 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5221 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5281 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5341 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagtttgcgg
     5401 ccgcaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     5461 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5521 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     5581 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5641 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5701 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5761 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5821 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5881 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5941 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6001 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6061 aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6121 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6181 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6241 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6301 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6361 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6421 ccgaaaagtg ccac
//
