LOCUS       sequence-333636-        7851 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP cDNA and p16 shRNA in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7851)
  AUTHORS   Smith JL, Lee LC, Read A, Li Q, Yu B, Lee CS, Luo J
  TITLE     One-step immortalization of primary human airway epithelial cells 
            capable of oncogenic transformation.
  JOURNAL   Cell Biosci. 2016 Nov 11;6:57. doi: 10.1186/s13578-016-0122-6. 
            eCollection 2016.
  PUBMED    27891214
REFERENCE   2  (bases 1 to 7851)
  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: "Cell 
            Biosci."; date: "2016-11-11"; volume: "6:57. doi"; pages: " 
            10.1186/s13578-016-0122-6. eCollection 2016"
COMMENT     Sequence Label: MSCVpic2neo-EGFP-p16 shRNA
FEATURES             Location/Qualifiers
     source          1..7851
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             987..1403
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1333..1355
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1371..1387
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     ncRNA           1443..1564
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           1592..1606
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           1634..1760
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     promoter        1827..2038
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2061..2070
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2067..2069,2070..2072,2073..2786)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDVLYK"
     primer_bind     complement(2112..2133)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2373..2392)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2720..2741
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2803..3376
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2826..3378
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2970..2987)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3197..3216
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3394..4197
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3457..3476)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4067..4086
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     LTR             4244..4758
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4920..4936
                     /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)."
     promoter        complement(join(4944..4950,4951..4968,4969..4974))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4989..5010
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5127..5144)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5298..5886)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5378..5397)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6057..6848,6849..6917))
                     /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     6680..6699
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6918..7022)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7090..7108
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7146..7168)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7268..7287
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7481..7503
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7631..7650
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(7850..7851,1..515)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaatta gatctctcga ctagggataa cagggtaatt
     1441 gtttgaatga ggcttcagta ctttacagaa tcgttgcctg cacatcttgg aaacacttgc
     1501 tgggattact tcttcaggtt aacccaacag aaggctcgag aaggtatatt gctgttgaca
     1561 gtgagcgact cgggaaactt agatcatcat agtgaagcca cagatgtatg atgatctaag
     1621 tttcccgagg tgcctactgc ctcggaattc aaggggctac tttaggagca attatcttgt
     1681 ttactaaaac tgaatacctt gctatctctt tgatacattt ttacaaagct gaattaaaat
     1741 ggtataaatt aaatcacttt tttcaattgg aagactaatg cgtttaaaca cgcggcgacg
     1801 cgtcgatggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa
     1861 gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg
     1921 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat
     1981 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt
     2041 gtcgtgacgc gggccggccc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg
     2101 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     2161 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     2221 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     2281 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2341 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     2401 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     2461 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     2521 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     2581 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     2641 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     2701 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     2761 tcggcatgga cgtgctgtac aagtaggcgg ccgccctgca ggcccctctc cctccccccc
     2821 ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt
     2881 attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt
     2941 cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa
     3001 tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac
     3061 cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg
     3121 tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt
     3181 tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca
     3241 gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tacacatgct ttacatgtgt
     3301 ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg
     3361 aaaaacacga tgataatatg gccacattaa ttaatgggat cggccattga acaagatgga
     3421 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     3481 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     3541 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg
     3601 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     3661 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     3721 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     3781 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     3841 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     3901 ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg
     3961 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     4021 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     4081 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     4141 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgaatc
     4201 gataaaataa aagattttat ttagtctcca gaaaaagggg ggaatgaaag accccacctg
     4261 taggtttggc aagctagctt aagtaacgcc attttgcaag gcatggaaaa tacataactg
     4321 agaatagaga agttcagatc aaggttagga acagagagac agcagaatat gggccaaaca
     4381 ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat ggtccccaga
     4441 tgcggtcccg ccctcagcag tttctagaga accatcagat gtttccaggg tgccccaagg
     4501 acctgaaatg accctgtgcc ttatttgaac taaccaatca gttcgcttct cgcttctgtt
     4561 cgcgcgcttc tgctccccga gctcaataaa agagcccaca acccctcact cggcgcgcca
     4621 gtcctccgat agactgcgtc gcccgggtac ccgtgtatcc aataaaccct cttgcagttg
     4681 catccgactt gtggtctcgc tgttccttgg gagggtctcc tctgagtgat tgactacccg
     4741 tcagcggggg tctttcatgg gtaacagttt cttgaagttg gagaacaaca ttctgagggt
     4801 aggagtcgaa tattaagtaa tcctgactca attagccact gttttgaatc cacatactcc
     4861 aatactcctg aaatagttca ttatggacag cgcagaaaga gctggggaga attgtgaaat
     4921 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4981 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     5041 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5101 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     5161 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5221 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5281 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5341 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5401 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5461 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5521 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5581 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5641 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5701 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     5761 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5821 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5881 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5941 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     6001 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     6061 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6121 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6181 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6241 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6301 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6361 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6421 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6481 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6541 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6601 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6661 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6721 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6781 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6841 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6901 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6961 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     7021 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     7081 acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt
     7141 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     7201 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt
     7261 aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg
     7321 cacagatgcg taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac
     7381 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
     7441 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
     7501 acgacggcgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     7561 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     7621 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     7681 tgccggccac gatgcgtccg gcgtagaggc gattagtcca atttgttaaa gacaggatat
     7741 cagtggtcca ggctctagtt ttgactcaac aatatcacca gctgaagcct atagagtacg
     7801 agccatagat aaaataaaag attttattta gtctccagaa aaagggggga a
//
