LOCUS       sequence-104376-        6639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression of EGFP-TEV-S-tag Mis12 for protein 
            localization and affinity purification (LAP).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6639)
  TITLE     Cheeseman Lab Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6639)
  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            "Unpublished"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: pIC65
FEATURES             Location/Qualifiers
     source          1..6639
                     /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(3055..3075)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3060..3389
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3240..3375
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3404..3451
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     LTR             3920..4513
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(4714..4731)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4885..5473)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4965..4984)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5651..6442,6443..6511))
                     /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     6274..6293
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6512..6616)
                     /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 cgagatgtct gtggatccaa tgacctacga
     2281 ggcccagttc tttggcttca cgccacaaac gtgcatgctt cggatctaca ttgcatttca
     2341 agactaccta tttgaagtga tgcaggccgt tgaacaggtt attctgaaga agctggatgg
     2401 catcccagac tgtgacatta gcccagtgca gattcgcaaa tgcacagaga agtttctttg
     2461 cttcatgaaa ggacattttg ataacctttt tagcaaaatg gagcaactgt ttttgcagct
     2521 gattttacgt attccctcaa acatcttgct tcctgaagat aaatgtaagg agacacctta
     2581 tagtgaggaa gattttcagc atctccagaa agaaattgaa cagttacagg agaagtacaa
     2641 gactgaatta tgtactaagc aggcccttct tgcagaatta gaagagcaaa aaattgttca
     2701 ggccaaactc aaacagacgt tgactttctt tgatgagctt cataatgttg gcagagatca
     2761 tgggactagt gattttaggg agagtttagt atccctggtt cagaactcca gaaaactaca
     2821 gaacattaga gacaatgtgg aaaaggaatc gaaacgactg aaaatatctt aaaagcttcg
     2881 aattctgcag tcgacggtac cgcgggcccg ggatccaccg gatctagata actgatcata
     2941 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
     3001 ctgaacctga aacataaaat gaatgcaatt cgccagcaca gtggtcgacc ctgtggaatg
     3061 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3121 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3181 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     3241 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     3301 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     3361 gcttttttgg aggcctaggc ttttgcaaaa agcttgagca cgtgttgaca attaatcatc
     3421 ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca tggccaagcc
     3481 tttgtctcaa gaagaatcca ccctcattga aagagcaacg gctacaatca acagcatccc
     3541 catctctgaa gactacagcg tcgccagcgc agctctctct agcgacggcc gcatcttcac
     3601 tggtgtcaat gtatatcatt ttactggggg accttgtgca gaactcgtgg tgctgggcac
     3661 tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag
     3721 gggcatcttg agcccctgcg gacggtgccg acaggtgctt ctcgatctgc atcctgggat
     3781 caaagccata gtgaggacag tgatggacag ccgacggcag ttgggattcg tgaattgctg
     3841 ccctctggtt atgtgtggga gggctaagca cttcatcgat aaaataaaag attttattta
     3901 gtctccagaa aaagggggga atgaaagacc ccacctgtag gtttggcaag ctagcttaag
     3961 taacgccatt ttgcaaggca tggaaaaata cataactgag aatagagaag ttcagatcaa
     4021 ggtcaggaac agatggaaca gctgaatatg ggccaaacag gatatctgtg gtaagcagtt
     4081 cctgccccgg ctcagggcca agaacagatg gaacagctga atatgggcca aacaggatat
     4141 ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggtccc cagatgcggt
     4201 ccagccctca gcagtttcta gagaaccatc agatgtttcc agggtgcccc aaggacctga
     4261 aatgaccctg tgccttattt gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg
     4321 cttctgctcc ccgagctcaa taaaagagcc cacaacccct cactcggggc gccagtcctc
     4381 cgattgactg agtcgcccgg gtacccgtgt atccaataaa ccctcttgca gttgcatccg
     4441 acttgtggtc tcgctgttcc ttgggagggt ctcctctgag tgattgacta cccgtcagcg
     4501 ggggtctttc atttccgact tgtggtctcg ctgccttggg agggtctcct ctgagtgatt
     4561 gactacccgt cagcgggggt cttcacatgc agcatgtatc aaaattaatt tggttttttt
     4621 tcttaagtat ttacattaaa tggccatagt tgcattaatg aatcggccaa cgcgcgggga
     4681 gaggcggttt gcgtattggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4741 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4801 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4861 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4921 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4981 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5041 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5101 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5161 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5221 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5281 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     5341 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5401 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5461 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5521 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     5581 tttaaattaa aaatgaagtt tgcggccgca aatcaatcta aagtatatat gagtaaactt
     5641 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5701 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5761 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5821 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5881 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5941 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6001 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6061 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6121 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6181 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6241 gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat agcagaactt
     6301 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6361 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6421 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6481 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6541 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6601 aaataggggt tccgcgcaca tttccccgaa aagtgccac
//
