LOCUS       sequence-104384-        6888 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression of EGFP-TEV-S-tag CENP-P for protein 
            localization and affinity purification (LAP).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6888)
  TITLE     Cheeseman Lab Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6888)
  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            "Unpublished"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: pIC146
FEATURES             Location/Qualifiers
     source          1..6888
                     /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(3304..3324)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3309..3638
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3489..3624
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3653..3700
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     LTR             4169..4762
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(4963..4980)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5134..5722)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5214..5233)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5900..6691,6692..6760))
                     /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     6523..6542
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6761..6865)
                     /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 cgagatggac gcagagctgg cagaggtgcg
     2281 cgccttgcaa gctgagatcg cggccctgcg gcgagcgtgt gaggacccac cggcgccctg
     2341 ggaagagaag tcccgagtcc aaaaatcttt tcaagccata caccaattca atttggaagg
     2401 atggaagtct tcaaaagatc tgaaaaatca gcttggacat ttagaatcag aactttcatt
     2461 tctaagtacg cttactggca tcaatataag aaatcactcc aagcagacag aagacctaac
     2521 aagcactgag atgacagaaa agagtattag aaaagttcta cagagacaca gattatcagg
     2581 aaattgccac atggttacat ttcaacttga atttcagatt ctggaaattc agaataagga
     2641 gagattatct tctgctgtta ctgacctcaa cataataatg gagcccacag aatgctcaga
     2701 attaagtgaa tttgtgtcta gagcagaaga gagaaaagat ctgttcatgt ttttccgaag
     2761 cctgcatttt tttgtggagt ggtttgaata tcgtaagcgc acgtttaaac atctcaagga
     2821 aaagtaccca gatgccgtgt acctctcgga ggggccctcc tcctgctcca tggggatccg
     2881 cagcgccagc cggccagggt ttgaattagt cattgtttgg aggatacaaa tagatgaaga
     2941 tgggaaggtt tttccaaagc tggatcttct caccaaagtc ccacagcgag ccctggagct
     3001 ggacaagaac agagccatag aaactgctcc tctcagcttc cgaaccctgg taggactgct
     3061 tggaatcgaa gctgctctgg aaagcctgat aaaatcgctt tgtgcagagg agaacaacta
     3121 gaagcttcga attctgcagt cgacggtacc gcgggcccgg gatccaccgg atctagataa
     3181 ctgatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca
     3241 cacctccccc tgaacctgaa acataaaatg aatgcaattc gccagcacag tggtcgaccc
     3301 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     3361 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     3421 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     3481 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     3541 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     3601 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gcttgagcac gtgttgacaa
     3661 ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat
     3721 ggccaagcct ttgtctcaag aagaatccac cctcattgaa agagcaacgg ctacaatcaa
     3781 cagcatcccc atctctgaag actacagcgt cgccagcgca gctctctcta gcgacggccg
     3841 catcttcact ggtgtcaatg tatatcattt tactggggga ccttgtgcag aactcgtggt
     3901 gctgggcact gctgctgctg cggcagctgg caacctgact tgtatcgtcg cgatcggaaa
     3961 tgagaacagg ggcatcttga gcccctgcgg acggtgccga caggtgcttc tcgatctgca
     4021 tcctgggatc aaagccatag tgaggacagt gatggacagc cgacggcagt tgggattcgt
     4081 gaattgctgc cctctggtta tgtgtgggag ggctaagcac ttcatcgata aaataaaaga
     4141 ttttatttag tctccagaaa aaggggggaa tgaaagaccc cacctgtagg tttggcaagc
     4201 tagcttaagt aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagagaagt
     4261 tcagatcaag gtcaggaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg
     4321 taagcagttc ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa
     4381 acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc
     4441 agatgcggtc cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca
     4501 aggacctgaa atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct
     4561 gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg
     4621 ccagtcctcc gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag
     4681 ttgcatccga cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac
     4741 ccgtcagcgg gggtctttca tttccgactt gtggtctcgc tgccttggga gggtctcctc
     4801 tgagtgattg actacccgtc agcgggggtc ttcacatgca gcatgtatca aaattaattt
     4861 ggtttttttt cttaagtatt tacattaaat ggccatagtt gcattaatga atcggccaac
     4921 gcgcggggag aggcggtttg cgtattggcg ctcttccgct tcctcgctca ctgactcgct
     4981 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5041 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5101 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5161 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5221 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5281 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5341 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5401 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5461 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5521 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     5581 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5641 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5701 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5761 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5821 ctagatcctt ttaaattaaa aatgaagttt gcggccgcaa atcaatctaa agtatatatg
     5881 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5941 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6001 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6061 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6121 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6181 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6241 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6301 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6361 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6421 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6481 gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg ggataatacc gcgccacata
     6541 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6601 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6661 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6721 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6781 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6841 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccac
//
