LOCUS       sequence-104382-        6831 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression of EGFP-TEV-S-tag CENP-K for protein 
            localization and affinity purification (LAP).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6831)
  TITLE     Cheeseman Lab Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6831)
  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            "Unpublished"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: pIC144
FEATURES             Location/Qualifiers
     source          1..6831
                     /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(3247..3267)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3252..3581
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3432..3567
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3596..3643
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     LTR             4112..4705
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(4906..4923)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5077..5665)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5157..5176)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5843..6634,6635..6703))
                     /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     6466..6485
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6704..6808)
                     /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 cgagatgaat caggaggatc tagatccgga
     2281 tagtactaca gatgtgggag atgttacaaa tactgaagaa gaacttatta gagaatgtga
     2341 agaaatgtgg aaagatatgg aagaatgtca gaataaatta tcacttattg gaactgaaac
     2401 actcaccgat tcaaatgctc agctatcatt gttaattatg caagtaaaat gtttaaccgc
     2461 tgaactcagt caatggcaga aaaaaacacc tgaaacaatt cccttgactg aagacgttct
     2521 cataacatta ggaaaagaag agttccaaaa gctgagacaa gatcttgaaa tggtactgtc
     2581 cactaaggag tcaaagaatg aaaagttaaa ggaagactta gaaagggaac aacggtggtt
     2641 ggatgaacag caacagataa tggaatctct taatgtacta cacagtgaat tgaaaaataa
     2701 ggttgaaaca ttttctgaat caagaatctt taatgaactg aaaactaaaa tgcttaatat
     2761 aaaagaatat aaggagaaac tcttgagtac cttgggcgag tttctagaag accattttcc
     2821 tctgcctgat agaagtgtta aaaagaaaaa gaaaaacatt caagaatcat ctgtaaacct
     2881 gataacactg catgaaatgt tagagattct tataaataga ttatttgatg ttccacatga
     2941 tccatatgtc aaaattagtg attccttttg gccaccttat gttgagctgc tgctgcgtaa
     3001 tggaattgcc ttgagacatc cagaagatcc aacccgaata agattagaag ctttccatca
     3061 gtaaaagctt cgaattctgc agtcgacggt accgcgggcc cgggatccac cggatctaga
     3121 taactgatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc
     3181 ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttcgccagca cagtggtcga
     3241 ccctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3301 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     3361 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     3421 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     3481 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     3541 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagcttgag cacgtgttga
     3601 caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac
     3661 catggccaag cctttgtctc aagaagaatc caccctcatt gaaagagcaa cggctacaat
     3721 caacagcatc cccatctctg aagactacag cgtcgccagc gcagctctct ctagcgacgg
     3781 ccgcatcttc actggtgtca atgtatatca ttttactggg ggaccttgtg cagaactcgt
     3841 ggtgctgggc actgctgctg ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg
     3901 aaatgagaac aggggcatct tgagcccctg cggacggtgc cgacaggtgc ttctcgatct
     3961 gcatcctggg atcaaagcca tagtgaggac agtgatggac agccgacggc agttgggatt
     4021 cgtgaattgc tgccctctgg ttatgtgtgg gagggctaag cacttcatcg ataaaataaa
     4081 agattttatt tagtctccag aaaaaggggg gaatgaaaga ccccacctgt aggtttggca
     4141 agctagctta agtaacgcca ttttgcaagg catggaaaaa tacataactg agaatagaga
     4201 agttcagatc aaggtcagga acagatggaa cagctgaata tgggccaaac aggatatctg
     4261 tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggaacagct gaatatgggc
     4321 caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga acagatggtc
     4381 cccagatgcg gtccagccct cagcagtttc tagagaacca tcagatgttt ccagggtgcc
     4441 ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc gcttctcgct
     4501 tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc ctcactcggg
     4561 gcgccagtcc tccgattgac tgagtcgccc gggtacccgt gtatccaata aaccctcttg
     4621 cagttgcatc cgacttgtgg tctcgctgtt ccttgggagg gtctcctctg agtgattgac
     4681 tacccgtcag cgggggtctt tcatttccga cttgtggtct cgctgccttg ggagggtctc
     4741 ctctgagtga ttgactaccc gtcagcgggg gtcttcacat gcagcatgta tcaaaattaa
     4801 tttggttttt tttcttaagt atttacatta aatggccata gttgcattaa tgaatcggcc
     4861 aacgcgcggg gagaggcggt ttgcgtattg gcgctcttcc gcttcctcgc tcactgactc
     4921 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4981 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5041 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5101 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5161 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5221 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     5281 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5341 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5401 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5461 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     5521 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5581 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5641 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5701 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5761 cacctagatc cttttaaatt aaaaatgaag tttgcggccg caaatcaatc taaagtatat
     5821 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5881 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5941 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     6001 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     6061 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     6121 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     6181 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     6241 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     6301 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     6361 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     6421 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac acgggataat accgcgccac
     6481 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6541 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6601 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6661 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6721 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6781 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca c
//
