LOCUS       sequence-135907-        8838 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression of mCherry-TEV-S-tag CENP-C for protein 
            localization and affinity purification (LAP).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8838)
  TITLE     Cheeseman Lab Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8838)
  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            "Unpublished"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: pKG123
FEATURES             Location/Qualifiers
     source          1..8838
                     /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             1391..2098
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1536..1554)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1804..1823
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2144..2164
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2177..2221
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(5253..5273)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5258..5587
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5438..5573
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        5602..5649
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5668..6066
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             6119..6712
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(6913..6930)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7084..7672)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7164..7183)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7850..8641,8642..8710))
                     /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     8473..8492
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8711..8815)
                     /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 ggataacatg gccatcatca aggagttcat
     1441 gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
     1501 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg
     1561 ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta
     1621 cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa
     1681 gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc
     1741 cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga
     1801 cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc
     1861 cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca
     1921 ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc
     1981 ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga
     2041 acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagtc
     2101 cggaggggat cggtggagct ccaccggtgg cggccgctct agagagaatc tttattttca
     2161 gggcgccgcc aaattcaaag aaaccgctgc tgctaaattc gaacgccagc acatggacag
     2221 cggaggtgga ggtactagtc tcgagatggc tgcgtccggt ctggatcatc tcaaaaatgg
     2281 ctacagaaga agattttgtc gaccttccag ggcacgtgac attaacacag agcaaggcca
     2341 gaatgttctg gaaatcttac aagactgttt tgaagaaaaa agtcttgcca atgattttag
     2401 tacaaattct acaaaatcag tgcctaattc aacacgcaaa ataaaagaca cttgtattca
     2461 gtcaccaagc aaagagtgcc agaaatcaca tccaaagtca gttccagttt cttcaaagaa
     2521 gaaagaagcc tctctacagt ttgttgtaga accaagtgaa gccacaaaca gatcagttca
     2581 ggcccatgaa gttcatcaga aaattctggc aactgatgtt agttccaaaa atacacctga
     2641 ctcgaaaaaa atatcaagta gaaacataaa tgatcatcac agtgaagctg atgaagaatt
     2701 ttacttatcc gttggctcac cttctgttct tttggatgca aaaacatctg tatcacaaaa
     2761 tgttattcca tctagtgcca aaaagagaga gacttacact tttgaaaatt cagtaaatat
     2821 gctgccttca agtacagagg tttcagttaa aaccaaaaaa aggttaaact ttgatgataa
     2881 agttatgtta aagaaaatag aaatagataa taaagtatca gatgaagagg ataaaacatc
     2941 ggaaggacaa gaaagaaaac catcaggatc atctcagaat agaatacgag attcagaata
     3001 tgaaattcaa cgacaagcta aaaaaagttt ttcaacattg tttttagaaa cagtaaaacg
     3061 aaaaagtgaa tccagtccca ttgttaggca tgcggcaact gctccacctc attcgtgtcc
     3121 tcccgatgat acgaagttga tagaggatga atttataatt gatgagtcgg atcaaagttt
     3181 tgccagtaga tcttggatta caataccaag aaaggcaggg tctctgaaac aacgcacaat
     3241 atccccggct gagagcactg cactctttca aggtagaaag tcaagagaaa agcatcataa
     3301 tatattacct aagactttgg caaatgacaa acattcccat aaacctcacc cagtagagac
     3361 atctcagccc tctgataaaa cagtactgga tacaagttat gctttgatag atgaaacagt
     3421 aaataattat agatctacaa aatatgaaat gtattccaag aatgcagaaa aaccatctag
     3481 aagcaaaagg actataaaac aaaaacagag aagaaaattc atggctaaac cagctgaaga
     3541 acagcttgat gtgggacagt ctaaagatga aaacatacat acatcacata ttacccaaga
     3601 cgaatttcaa agaaattcag acagaaatat ggaagagcat gaagagatgg gaaatgattg
     3661 tgtttccaaa aaacagatgc cacctgtggg aagcaagaaa agtagcacta gaaaagataa
     3721 ggaagaatct aaaaagaagc gcttttccag tgagtccaag aacaaacttg tacctgaaga
     3781 agtgacttca actgtcacga aaagtcgaag aatttccagg cgtccatctg attggtgggt
     3841 ggtaaaatca gaggagagtc ctgtttatag caattcttca gtaagaaatg aattaccaat
     3901 gcatcacaat agtagccgaa aatctactaa gaaaacaaat cagtcatcta agaatattag
     3961 gaaaaaaact attccactta aaaggcagaa gacagcaact aaaggcaacc aaagagtaca
     4021 gaagttttta aatgctgaag gttctggagg tatcgttggt catgatgaaa tttccagatg
     4081 ttcactgagt gagccattgg aaagtgatga ggcagacttg gctaagaaga aaaatcttga
     4141 ttgttctaga tctacaagaa gctcaaagaa tgaagataac attatgactg cacagaatgt
     4201 tcccctaaag cctcagacca gtggatatac atgtaatata ccaacagagt caaacttgga
     4261 ttctggagag cataagactt cagttttaga ggaaagtgga ccttccaggc tcaataataa
     4321 ttatttaatg tctggaaaga atgatgtgga tgatgaggaa gttcatggaa gttcagatga
     4381 ctcaaaacaa tctaaagtga taccaaagaa cagaatccat cacaaactag tattgccctc
     4441 caacacacca aatgttcgca ggaccaagag aacacgtttg aaacctttgg agtactggcg
     4501 aggagagcga atagattatc aaggaaggcc atcaggagga ttcgtgatta gtggagtact
     4561 atctccagac acaatatcgt ctaaaaggaa ggcaaaagaa aatattggaa aagtcaacaa
     4621 aaaatctaat aagaaaagga tctgtcttga taacgatgaa agaaagacta acttaatggt
     4681 aaatctaggt atacctcttg gagatccttt gcagccaacg agggtaaagg acccagaaac
     4741 aagagagatt attctcatgg atcttgtaag gccacaagat acatatcaat tttttgttaa
     4801 gcatggtgag ttgaaggtat acaagacatt ggatacaccc tttttttcta ctgggaaatt
     4861 gatattagga ccacaagaag aaaagggaaa gcagcatgtt ggccaggata tattggtttt
     4921 ttatgttaac tttggtgacc ttttgtgtac tttacatgaa acaccttata tattaagtac
     4981 tggggattcg ttctatgttc cttcaggtaa ctattataac atcaaaaatc tccggaatga
     5041 ggaaagtgtt cttcttttta ctcagataaa aagatgagaa ttctgcagtc gacggtaccg
     5101 cgggcccggg atccaccgga tctagataac tgatcataat cagccatacc acatttgtag
     5161 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     5221 atgcaattcg ccagcacagt ggtcgaccct gtggaatgtg tgtcagttag ggtgtggaaa
     5281 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     5341 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     5401 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     5461 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     5521 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     5581 ttgcaaaaag cttgagcacg tgttgacaat taatcatcgg catagtatat cggcatagta
     5641 taatacgaca aggtgaggaa ctaaaccatg gccaagcctt tgtctcaaga agaatccacc
     5701 ctcattgaaa gagcaacggc tacaatcaac agcatcccca tctctgaaga ctacagcgtc
     5761 gccagcgcag ctctctctag cgacggccgc atcttcactg gtgtcaatgt atatcatttt
     5821 actgggggac cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc ggcagctggc
     5881 aacctgactt gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag cccctgcgga
     5941 cggtgccgac aggtgcttct cgatctgcat cctgggatca aagccatagt gaaggacagt
     6001 gatggacagc cgacggcagt tgggattcgt gaattgctgc cctctggtta tgtgtgggag
     6061 ggctaagcac ttcatcgata aaataaaaga ttttatttag tctccagaaa aaggggggaa
     6121 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
     6181 ggaaaaatac ataactgaga atagagaagt tcagatcaag gtcaggaaca gatggaacag
     6241 ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
     6301 gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc
     6361 ccggctcagg gccaagaaca gatggtcccc agatgcggtc cagccctcag cagtttctag
     6421 agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg
     6481 aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat
     6541 aaaagagccc acaacccctc actcggggcg ccagtcctcc gattgactga gtcgcccggg
     6601 tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct
     6661 tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca tttccgactt
     6721 gtggtctcgc tgccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc
     6781 ttcacatgca gcatgtatca aaattaattt ggtttttttt cttaagtatt tacattaaat
     6841 ggccatagtt gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattggcg
     6901 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     6961 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     7021 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     7081 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     7141 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     7201 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     7261 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     7321 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     7381 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     7441 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     7501 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     7561 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     7621 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     7681 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     7741 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     7801 gcggccgcaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     7861 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     7921 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     7981 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     8041 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     8101 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     8161 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     8221 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     8281 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     8341 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     8401 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     8461 cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     8521 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     8581 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     8641 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     8701 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     8761 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     8821 ttccccgaaa agtgccac
//
