LOCUS       sequence-363717-        7165 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7165)
  AUTHORS   Hatterschide J, Castagnino P, Kim HW, Sperry SM, Montone KT, Basu D,
            White EA
  TITLE     YAP1 activation by human papillomavirus E7 promotes basal cell 
            identity in squamous epithelia.
  JOURNAL   Elife. 2022 Feb 16;11. pii: 75466. doi: 10.7554/eLife.75466.
  PUBMED    35170430
REFERENCE   2  (bases 1 to 7165)
  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: "Elife."; 
            date: "2022-02-16"; volume: "11. pii: 75466. doi"; pages: " 
            10.7554/eLife.75466"
COMMENT     Sequence Label: p8130 MSCV GFP Puro C-FlagHA
FEATURES             Location/Qualifiers
     source          1..7165
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(23..45)
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     misc_feature    458..799
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     LTR             863..1379
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     primer_bind     complement(1579..1598)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(1726..1748)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1942..1961)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2061..2083
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2121..2139)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2207..2311
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2312..2380,2381..3172)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2530..2549)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3343..3931
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3832..3851
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4085..4102
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4219..4240
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4255..4260,4261..4278,4279..4285)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4293..4309
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     LTR             4471..4985
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     CDS             complement(5085..5684)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5168..5188)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     5665..5684
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(5705..6204)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5753..5772)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     6146..6169
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     CDS             complement(6217..6243)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             complement(6217..6243)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(6259..6282)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    6314..6338
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(6337..7050,7051..7053,7054..7056))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(6340..7050,7051..7053,7054..7056))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6382..6403)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     6731..6750
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6990..7011
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(7056..7080)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(7109..7128)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7110..7128)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(7140..7165,1..391))
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(join(7156..7165,1..7))
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
ORIGIN
        1 gataaaggga ggatcgaggc ggggtcgaac gaggaggttc aagggggaga gacggggcgg
       61 atggaggaag aggaggcgga ggcttagggt gtacaaaggg cttgacccag ggaggggggt
      121 caaaagccaa ggcttcccag gtcacgatgt aggggacctg gtctgggtgt ccatgcgggc
      181 caggtgaaaa gaccttgatc ttaacctggg tgatgaggtc tcggttaaag gtgccgtctc
      241 gcggccatcc gacgttaaag gttggccatt ctgcagagca gaaggtaacc caacgtctct
      301 tcttgacatc taccgactgg ttgtgagcga tccgctcgac atctttccag tgacctaagg
      361 tcaaacttaa gggagtggta acagtctggc cctaattttc agacaaatac agaaacacag
      421 tcagacagag acaacacaga acgatgctgc agcgctgcag cagacaagac gcgcggcttc
      481 ggttccaaac cgaaagcaaa aattcagacg gaggcgggaa ctgttttagg ttctcgtctc
      541 ctaccagaac cacatatcct gacggggtcg gattccacat cgactccctt cctcaggtcg
      601 ggccacaaaa acggccccca aagtccctgg gacgtctccc agggttgcgg ccgggtgttc
      661 agaactcgtc agttccacca cgggtccgcc agatacagag ctagttagct aactagtaca
      721 gacgcaggcg caaacattag atgccggcac aaacacgcac aaagacagag acagacacga
      781 aacgaccgct ggccagctta cctcccggcg ggggggtcgg tggtccctgg gcaggggtct
      841 ccaaatctcg gtggaacctc caaatgaaag acccccgagg tgggcagtca atcaatctga
      901 ggagaccctc ccaaggatca gcgagtccac gattcggatg caaacagcaa gaggctttat
      961 tgggaatacg ggtacccggg cgacgcagtc tatcggagga ctggcgcgcc gagtgagggg
     1021 ttgtgggctc ttttattgag ctcggggagc agaagcgcgc gaacagaagc gagaagcgaa
     1081 ctgattggtt agttcaaata aggcacaggg tcatttcagg tccttggggc accctggaaa
     1141 catctgatgg ttctctagaa actgctgagg gcgggaccgc atctggggac catctgttct
     1201 tggccctgag ccggggcagg aactgcttac cacagatatc ctgtttggcc catattctgc
     1261 tgtctctctg ttcctaacct tgatctgaac ttctctattc tcagttatgt attttccatg
     1321 ccttgcaaaa tggcgttact taagctagct tgccaaacct acaggtgggg tctttcattc
     1381 cccccttttt ctggagacta aataaaatct tttattttat ctatggctcg tactctatag
     1441 gcttcagctg gtgatattgt tgagtcaaaa ctagagcctg gaccactgat atcctgtctt
     1501 taacaaattg gactaatcgc ctctacgccg gacgcatcgt ggccggcatc accggcgcca
     1561 caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc
     1621 acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg
     1681 gactgttggg cgccatctcc ttgcatgcac cattccttgc gccgtcgttt tacaacgtcg
     1741 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     1801 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     1861 gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     1921 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     1981 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     2041 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     2101 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     2161 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     2221 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     2281 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     2341 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     2401 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     2461 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     2521 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     2581 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     2641 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     2701 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     2761 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     2821 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     2881 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     2941 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     3001 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     3061 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     3121 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     3181 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     3241 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     3301 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     3361 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     3421 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     3481 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     3541 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     3601 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     3661 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     3721 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     3781 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccaa ggggaaacgc
     3841 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     3901 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     3961 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     4021 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     4081 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     4141 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     4201 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     4261 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacaattc
     4321 tccccagctc tttctgcgct gtccataatg aactatttca ggagtattgg agtatgtgga
     4381 ttcaaaacag tggctaattg agtcaggatt acttaatatt cgactcctac cctcagaatg
     4441 ttgttctcca acttcaagaa actgttaccc atgaaagacc cccgctgacg ggtagtcaat
     4501 cactcagagg agaccctccc aaggaacagc gagaccacaa gtcggatgca actgcaagag
     4561 ggtttattgg atacacgggt acccgggcga cgcagtctat cggaggactg gcgcgccgag
     4621 tgaggggttg tgggctcttt tattgagctc ggggagcaga agcgcgcgaa cagaagcgag
     4681 aagcgaactg attggttagt tcaaataagg cacagggtca tttcaggtcc ttggggcacc
     4741 ctggaaacat ctgatggttc tctagaaact gctgagggcg ggaccgcatc tggggaccat
     4801 ctgttcttgg ccctgagccg gggcaggaac tgcttaccac agatatcctg tttggcccat
     4861 attctgctgt ctctctgttc ctaaccttga tctgaacttc tctattctca gttatgtatt
     4921 ttccatgcct tgcaaaatgg cgttacttaa gctagcttgc caaacctaca ggtggggtct
     4981 ttcattcccc cctttttctg gagactaaat aaaatctttt attttatcga tgcatggggt
     5041 cgtgcgctcc tttcggtcgg gcgctgcggg tcgtggggcg ggcgtcaggc accgggcttg
     5101 cgggtcatgc accaggtgcg cggtccttcg ggcacctcga cgtcggcggt gacggtgaag
     5161 ccgagccgct cgtagaaggg gaggttgcgg ggcgcggagg tctccaggaa ggcgggcacc
     5221 ccggcgcgct cggccgcctc cactccgggg agcacgacgg cgctgcccag acccttgccc
     5281 tggtggtcgg gcgagacgcc gacggtggcc aggaaccacg cgggctcctt gggccggtgc
     5341 ggcgccagga ggccttccat ctgttgctgc gcggccagcc gggaaccgct caactcggcc
     5401 atgcgcgggc cgatctcggc gaacaccgcc cccgcttcga cgctctccgg cgtggtccag
     5461 accgccaccg cggcgccgtc gtccgcgacc cacaccttgc cgatgtcgag cccgacgcgc
     5521 gtgaggaaga gttcttgcag ctcggtgacc cgctcgatgt ggcggtccgg atcgacggtg
     5581 tggcgcgtgg cggggtagtc ggcgaacgcg gcggcgaggg tgcgtacggc cctggggacg
     5641 tcgtcgcggg tggcgaggcg caccgtgggc ttgtactcgg tcatggtaag cttgggctgc
     5701 aggtcgaaag gcccggagat gaggaagagg agaacagcgc ggcagacgtg cgcttttgaa
     5761 gcgtgcagaa tgccgggcct ccggaggacc ttcgggcgcc cgccccgccc ctgagcccgc
     5821 ccctgagccc gcccccggac ccaccccttc ccagcctctg agcccagaaa gcgaaggagc
     5881 aaagctgcta ttggccgctg ccccaaaggc ctacccgctt ccattgctca gcggtgctgt
     5941 ccatctgcac gagactagtg agacgtgcta cttccatttg tcacgtcctg cacgacgcga
     6001 gctgcggggc gggggggaac ttcctgacta ggggaggagt agaaggtggc gcgaaggggc
     6061 caccaaagaa cggagccggt tggcgcctac cggtggatgt ggaatgtgtg cgaggccaga
     6121 ggccacttgt gtagcgccaa gtgcccagcg gggctgctaa agcgcatgct ccagactgcc
     6181 ttgggaaaag cgcctcccct acccggtaga attctaggcg tagtcgggca cgtcgtaggg
     6241 gtatcctcca gcggccgact tgtcatcgtc gtccttgtag tcctcgagtt cgaaccgcgg
     6301 gccctctaga tcaaccactt tgtacaagaa agttggtcac ttgtacagct cgtccatgcc
     6361 gagagtgatc ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt
     6421 ggggtctttg ctcagggcgg actgggtgct caggtagtgg ttgtcgggca gcagcacggg
     6481 gccgtcgccg atgggggtgt tctgctggta gtggtcggcg agctgcacgc tgccgtcctc
     6541 gatgttgtgg cggatcttga agttcacctt gatgccgttc ttctgcttgt cggccatgat
     6601 atagacgttg tggctgttgt agttgtactc cagcttgtgc cccaggatgt tgccgtcctc
     6661 cttgaagtcg atgcccttca gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac
     6721 ctcggcgcgg gtcttgtagt tgccgtcgtc cttgaagaag atggtgcgct cctggacgta
     6781 gccttcgggc atggcggact tgaagaagtc gtgctgcttc atgtggtcgg ggtagcggct
     6841 gaagcactgc acgccgtagg tcagggtggt cacgagggtg ggccagggca cgggcagctt
     6901 gccggtggtg cagatgaact tcagggtcag cttgccgtag gtggcatcgc cctcgccctc
     6961 gccggacacg ctgaacttgt ggccgtttac gtcgccgtcc agctcgacca ggatgggcac
     7021 caccccggtg aacagctcct cgcccttgct caccatgcca acttttttgt acaaacttgt
     7081 tgatagctta actagccagc ttgggtctcc ctatagtgag tcgtattaga tctaattccg
     7141 gcgcctagag aaggagtgag ggctg
//
