LOCUS       sequence-134682-        6924 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression of EGFP-TEV-S-tag CENP-O for protein 
            localization and affinity purification (LAP).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6924)
  TITLE     Cheeseman Lab Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6924)
  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            "Unpublished"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: pIC145
FEATURES             Location/Qualifiers
     source          1..6924
                     /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(3340..3360)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3345..3674
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3525..3660
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3689..3736
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     LTR             4205..4798
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(4999..5016)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5170..5758)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5250..5269)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5936..6727,6728..6796))
                     /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     6559..6578
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6797..6901)
                     /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 cgagatggag caggcgaacc ctttacgtcc
     2281 agatggcgag tccaaaggag gtgttttagc tcacttggaa aggctagaga cccaagtgag
     2341 cagatcccgt aaacagtctg aagagctgca gagcgtgcag gcccaggaag gtgctcttgg
     2401 aaccaagatt cataaactaa ggcgtctgcg agatgagctg agggctgtgg tgcggcaccg
     2461 gcgagccagc gtgaaagcat gtattgccaa tgtagaaccc aaccaaacag tggagatcaa
     2521 tgagcaagaa gcattggaag agaaattgga aaatgtgaaa gccattctgc aggcatatca
     2581 ttttacaggc ctcagtggta aactgaccag ccgaggagtt tgtgtctgca tcagtactgc
     2641 ttttgagggg aacctattgg attcctattt tgtggacctt gtcatacaga aaccactccg
     2701 gatacatcac cattcagtcc cagtcttcat tcccctggaa gagatagctg caaaatattt
     2761 acagaccaac atccagcact tcctgttcag tctctgcgag tacctgaatg cttactctgg
     2821 gaggaagtac caggcagacc ggcttcagag tgactttgca gccctcctga ctgggccctt
     2881 gcagagaaac ccactgtgta acttgctgtc atttacttac aaactggatc cagggggtca
     2941 gtccttcccg ttctgtgcta gattgctgta taaggacctc acagcaactc ttcccactga
     3001 cgtcaccgtg acatgtcaag gagtggaagt attatccact tcatgggagg agcaacgagc
     3061 atcccatgaa actctgttct gtacgaagcc cttgcatcaa gtgtttgcct catttacaag
     3121 aaaaggagaa aagttggata tgagtctggt ctcctaaaag cttcgaattc tgcagtcgac
     3181 ggtaccgcgg gcccgggatc caccggatct agataactga tcataatcag ccataccaca
     3241 tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat
     3301 aaaatgaatg caattcgcca gcacagtggt cgaccctgtg gaatgtgtgt cagttagggt
     3361 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3421 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3481 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     3541 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     3601 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     3661 taggcttttg caaaaagctt gagcacgtgt tgacaattaa tcatcggcat agtatatcgg
     3721 catagtataa tacgacaagg tgaggaacta aaccatggcc aagcctttgt ctcaagaaga
     3781 atccaccctc attgaaagag caacggctac aatcaacagc atccccatct ctgaagacta
     3841 cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtg tcaatgtata
     3901 tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc
     3961 agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggca tcttgagccc
     4021 ctgcggacgg tgccgacagg tgcttctcga tctgcatcct gggatcaaag ccatagtgag
     4081 gacagtgatg gacagccgac ggcagttggg attcgtgaat tgctgccctc tggttatgtg
     4141 tgggagggct aagcacttca tcgataaaat aaaagatttt atttagtctc cagaaaaagg
     4201 ggggaatgaa agaccccacc tgtaggtttg gcaagctagc ttaagtaacg ccattttgca
     4261 aggcatggaa aaatacataa ctgagaatag agaagttcag atcaaggtca ggaacagatg
     4321 gaacagctga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag
     4381 ggccaagaac agatggaaca gctgaatatg ggccaaacag gatatctgtg gtaagcagtt
     4441 cctgccccgg ctcagggcca agaacagatg gtccccagat gcggtccagc cctcagcagt
     4501 ttctagagaa ccatcagatg tttccagggt gccccaagga cctgaaatga ccctgtgcct
     4561 tatttgaact aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct gctccccgag
     4621 ctcaataaaa gagcccacaa cccctcactc ggggcgccag tcctccgatt gactgagtcg
     4681 cccgggtacc cgtgtatcca ataaaccctc ttgcagttgc atccgacttg tggtctcgct
     4741 gttccttggg agggtctcct ctgagtgatt gactacccgt cagcgggggt ctttcatttc
     4801 cgacttgtgg tctcgctgcc ttgggagggt ctcctctgag tgattgacta cccgtcagcg
     4861 ggggtcttca catgcagcat gtatcaaaat taatttggtt ttttttctta agtatttaca
     4921 ttaaatggcc atagttgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4981 ttggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     5041 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     5101 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     5161 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5221 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     5281 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     5341 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     5401 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5461 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5521 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5581 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     5641 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5701 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5761 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5821 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5881 aagtttgcgg ccgcaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5941 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6001 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6061 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6121 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6181 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6241 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6301 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6361 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6421 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6481 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6541 gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6601 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6661 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6721 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6781 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6841 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6901 gcacatttcc ccgaaaagtg ccac
//
