LOCUS       sequence-104389-        6282 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression of EGFP-TEV-S-tag CENP-W for protein 
            localization and affinity purification (LAP).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6282)
  TITLE     Cheeseman Lab Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6282)
  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            "Unpublished"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: pKG136
FEATURES             Location/Qualifiers
     source          1..6282
                     /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(2697..2717)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2702..3031
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2882..3017
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3046..3093
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3112..3510
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             3563..4156
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(4357..4374)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4528..5116)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4608..4627)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5294..6085,6086..6154))
                     /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     5917..5936
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6155..6259)
                     /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 cgagatggca ctgtctacga ttgtgagtca
     2281 gcgtaaacaa atcaaacgta aagcccctcg tggtttcctg aaacgtgtgt ttaaacgtaa
     2341 aaaacctcag ctgcgcctgg aaaaatcagg tgatctgctg gttcatctga attgcctgtt
     2401 attcgtccac cgtctggcag aagaatcccg tactaatgcc tgcgcttcga aatgtcgtgt
     2461 catcaacaaa gaacatgtgc tggctgccgc taaagtcatt ctgaaaaaat cccgtggctg
     2521 agaattctgc agtcgacggt accgcgggcc cgggatccac cggatctaga taactgatca
     2581 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     2641 ccctgaacct gaaacataaa atgaatgcaa ttcgccagca cagtggtcga ccctgtggaa
     2701 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     2761 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     2821 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     2881 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     2941 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     3001 aggctttttt ggaggcctag gcttttgcaa aaagcttgag cacgtgttga caattaatca
     3061 tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag
     3121 cctttgtctc aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc
     3181 cccatctctg aagactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc
     3241 actggtgtca atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc
     3301 actgctgctg ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac
     3361 aggggcatct tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg
     3421 atcaaagcca tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg
     3481 ctgccctctg gttatgtgtg ggagggctaa gcacttcatc gataaaataa aagattttat
     3541 ttagtctcca gaaaaagggg ggaatgaaag accccacctg taggtttggc aagctagctt
     3601 aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagag aagttcagat
     3661 caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca
     3721 gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg ccaaacagga
     3781 tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc
     3841 ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc
     3901 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc
     3961 gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc
     4021 ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat
     4081 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca
     4141 gcgggggtct ttcatttccg acttgtggtc tcgctgcctt gggagggtct cctctgagtg
     4201 attgactacc cgtcagcggg ggtcttcaca tgcagcatgt atcaaaatta atttggtttt
     4261 ttttcttaag tatttacatt aaatggccat agttgcatta atgaatcggc caacgcgcgg
     4321 ggagaggcgg tttgcgtatt ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4381 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4441 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4501 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4561 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4621 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4681 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4741 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4801 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4861 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4921 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     4981 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5041 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5101 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5161 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5221 ccttttaaat taaaaatgaa gtttgcggcc gcaaatcaat ctaaagtata tatgagtaaa
     5281 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5341 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5401 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5461 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5521 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5581 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     5641 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5701 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5761 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5821 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5881 ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca catagcagaa
     5941 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6001 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6061 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6121 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6181 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6241 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc ac
//
