LOCUS       sequence-134679-        6793 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 6793)
  TITLE     Cheeseman Lab Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6793)
  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            "Unpublished"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..6793
                     /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(3209..3229)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3214..3543
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3394..3529
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3558..3605
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     LTR             4074..4667
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(4868..4885)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5039..5627)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5119..5138)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5805..6596,6597..6665))
                     /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     6428..6447
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6666..6770)
                     /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 caggaggatt 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 gtaaggatcc accggatcta gataactgat cataatcagc cataccacat ttgtagaggt
     3121 tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc
     3181 aattcgccag cacagtggtc gaccctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     3241 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     3301 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3361 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     3421 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     3481 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     3541 aaaaagcttg agcacgtgtt gacaattaat catcggcata gtatatcggc atagtataat
     3601 acgacaaggt gaggaactaa accatggcca agcctttgtc tcaagaagaa tccaccctca
     3661 ttgaaagagc aacggctaca atcaacagca tccccatctc tgaagactac agcgtcgcca
     3721 gcgcagctct ctctagcgac ggccgcatct tcactggtgt caatgtatat cattttactg
     3781 ggggaccttg tgcagaactc gtggtgctgg gcactgctgc tgctgcggca gctggcaacc
     3841 tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc tgcggacggt
     3901 gccgacaggt gcttctcgat ctgcatcctg ggatcaaagc catagtgagg acagtgatgg
     3961 acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt gggagggcta
     4021 agcacttcat cgataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa
     4081 gaccccacct gtaggtttgg caagctagct taagtaacgc cattttgcaa ggcatggaaa
     4141 aatacataac tgagaataga gaagttcaga tcaaggtcag gaacagatgg aacagctgaa
     4201 tatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca
     4261 gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc
     4321 tcagggccaa gaacagatgg tccccagatg cggtccagcc ctcagcagtt tctagagaac
     4381 catcagatgt ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta
     4441 accaatcagt tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag
     4501 agcccacaac ccctcactcg gggcgccagt cctccgattg actgagtcgc ccgggtaccc
     4561 gtgtatccaa taaaccctct tgcagttgca tccgacttgt ggtctcgctg ttccttggga
     4621 gggtctcctc tgagtgattg actacccgtc agcgggggtc tttcatttcc gacttgtggt
     4681 ctcgctgcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtcttcac
     4741 atgcagcatg tatcaaaatt aatttggttt tttttcttaa gtatttacat taaatggcca
     4801 tagttgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tggcgctctt
     4861 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4921 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4981 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     5041 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5101 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5161 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     5221 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5281 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     5341 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     5401 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5461 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5521 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5581 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5641 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5701 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agtttgcggc
     5761 cgcaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     5821 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5881 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5941 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6001 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6061 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6121 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6181 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6241 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6301 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6361 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6421 acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6481 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6541 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6601 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6661 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6721 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6781 cgaaaagtgc cac
//
