LOCUS       sequence-104390-        6909 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression of EGFP-TEV-S-tag FANC-A for protein 
            localization and affinity purification (LAP).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6909)
  TITLE     Cheeseman Lab Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6909)
  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            "Unpublished"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: pKG186
FEATURES             Location/Qualifiers
     source          1..6909
                     /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(3324..3344)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3329..3658
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3509..3644
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3673..3720
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3739..4137
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             4190..4783
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(4984..5001)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5155..5743)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5235..5254)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5921..6712,6713..6781))
                     /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     6544..6563
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6782..6886)
                     /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 cgagatgtcc gactcgtggg tcccgaactc
     2281 cgcctcgggc caggacccag ggggccgccg gagggcctgg gccgagctgc tggcgggaag
     2341 ggtcaagagg gaaaaatata atcctgaacg ggcacagaaa ttaaaggaat cagctgtgcg
     2401 cctcctgcga agccatcagg acctgaatgc ccttttgctt gaggtagaag gtccactgtg
     2461 taaaaaattg tctctcagca aagtgattga ctgtgacagt tctgaggcct atgctaatca
     2521 ttctagttca tttataggct ctgctttgca ggatcaagcc tcaaggctgg gggttcccgt
     2581 gggtattctc tcagccggga tggttgcctc tagcgtggga cagatctgca cggctccagc
     2641 ggagaccagt caccctgtgc tgctgactgt ggagcagaga aagaagctgt cttccctgtt
     2701 agagtttgct cagtatttat tggcacacag tatgttctcc cgtctttcct tctgtcaaga
     2761 attatggaaa atacagagtt ctttgttgct tgaagcggtg tggcatcttc acgtacaagg
     2821 cattgtgagc ctgcaagagc tgctggaaag ccatcccgac atgcatgctg tgggatcgtg
     2881 gctcttcagg aatctgtgct gcctttgtga acagatggaa gcatcctgcc agcatgctga
     2941 cgtcgccagg gccatgcttt ctgattttgt tcaaatgttt gttttgaggg gatttcagaa
     3001 aaactcagat ctgagaagaa ctgtggagcc tgaaaaaatg ccgcaggtcg cggttgatgt
     3061 actgcagaga atgctgattt ttgcacttga cgctttggct gctggagtac aggaggagtc
     3121 ctccactcac aagatcgtga ggtgctgaga attctgcagt cgacggtacc gcgggcccgg
     3181 gatccaccgg atctagataa ctgatcataa tcagccatac cacatttgta gaggttttac
     3241 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattc
     3301 gccagcacag tggtcgaccc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     3361 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     3421 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3481 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     3541 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     3601 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     3661 gcttgagcac gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac
     3721 aaggtgagga actaaaccat ggccaagcct ttgtctcaag aagaatccac cctcattgaa
     3781 agagcaacgg ctacaatcaa cagcatcccc atctctgaag actacagcgt cgccagcgca
     3841 gctctctcta gcgacggccg catcttcact ggtgtcaatg tatatcattt tactggggga
     3901 ccttgtgcag aactcgtggt gctgggcact gctgctgctg cggcagctgg caacctgact
     3961 tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg acggtgccga
     4021 caggtgcttc tcgatctgca tcctgggatc aaagccatag tgaaggacag tgatggacag
     4081 ccgacggcag ttgggattcg tgaattgctg ccctctggtt atgtgtggga gggctaagca
     4141 cttcatcgat aaaataaaag attttattta gtctccagaa aaagggggga atgaaagacc
     4201 ccacctgtag gtttggcaag ctagcttaag taacgccatt ttgcaaggca tggaaaaata
     4261 cataactgag aatagagaag ttcagatcaa ggtcaggaac agatggaaca gctgaatatg
     4321 ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg
     4381 gaacagctga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag
     4441 ggccaagaac agatggtccc cagatgcggt ccagccctca gcagtttcta gagaaccatc
     4501 agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca
     4561 atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc
     4621 cacaacccct cactcggggc gccagtcctc cgattgactg agtcgcccgg gtacccgtgt
     4681 atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc ttgggagggt
     4741 ctcctctgag tgattgacta cccgtcagcg ggggtctttc atttccgact tgtggtctcg
     4801 ctgccttggg agggtctcct ctgagtgatt gactacccgt cagcgggggt cttcacatgc
     4861 agcatgtatc aaaattaatt tggttttttt tcttaagtat ttacattaaa tggccatagt
     4921 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggc gctcttccgc
     4981 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     5041 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     5101 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     5161 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5221 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5281 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5341 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     5401 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5461 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5521 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5581 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5641 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     5701 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     5761 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     5821 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt tgcggccgca
     5881 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5941 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6001 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6061 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     6121 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6181 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     6241 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     6301 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     6361 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6421 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6481 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac
     6541 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6601 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6661 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6721 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6781 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6841 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6901 aagtgccac
//
