LOCUS       sequence-245707-        7749 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive, ubiquitous GFP expression. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7749)
  AUTHORS   Jacob A, Vedaie M, Roberts DA, Thomas DC, Villacorta-Martin C, 
            Alysandratos KD, Hawkins F, Kotton DN
  TITLE     Derivation of self-renewing lung alveolar epithelial type II cells 
            from human pluripotent stem cells.
  JOURNAL   Nat Protoc. 2019 Dec;14(12):3303-3332. doi: 
            10.1038/s41596-019-0220-0. Epub 2019 Nov 15.
  PUBMED    31732721
REFERENCE   2  (bases 1 to 7749)
  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"; doi: 
            "10.1038/s41596-019-0220-0"; journalName: "Nat Protoc"; date: 
            "2019-12"; volume: "14"; issue: "12"; pages: "3303-3332"
COMMENT     Sequence Label: pHAGE-EF1aL-eGFP-W
FEATURES             Location/Qualifiers
     source          1..7749
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2058..2175
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2223..3401
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2454..3392
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3349..3369
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3406..3415
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3412..3414,3415..3417,3418..4131)
                     /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             join(3412..3414,3415..3417,3418..4128)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3457..3478)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3718..3737)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4065..4086
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    4139..4727
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4192..4212)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4610..4621)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4802..5035
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(5154..5172)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5240..5344
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5345..5413,5414..6205)
                     /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     complement(5563..5582)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6376..6964
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6865..6884
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7118..7135
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7375..7396
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7411..7416,7417..7434,7435..7441)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7449..7465
                     /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)."
     primer_bind     7454..7476
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7473..7489
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7473..7489
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7539..7558
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttcacaaatg
     2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagtcgt
     2221 gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg
     2281 ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa
     2341 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg
     2401 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg
     2461 ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa
     2521 ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt
     2581 gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct
     2641 ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg
     2701 ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct
     2761 ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg
     2821 ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc
     2881 gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc
     2941 ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac
     3001 cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga
     3061 ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc
     3121 cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg
     3181 attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga
     3241 tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt
     3301 aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga
     3361 cagtggttca aagttttttt cttccatttc aggtgtcgtg aagcggccgc catggtgagc
     3421 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     3481 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     3541 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     3601 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     3661 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     3721 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     3781 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     3841 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     3901 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     3961 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     4021 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     4081 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta aggatcctaa
     4141 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     4201 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     4261 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     4321 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     4381 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     4441 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     4501 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
     4561 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     4621 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     4681 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcctg agatccttta
     4741 agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga
     4801 ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct
     4861 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     4921 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     4981 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtagt
     5041 agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt
     5101 gagaggcccg ggttaattaa ggaaagggct agatcattct tgaagacgaa agggcctcgt
     5161 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     5221 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5281 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5341 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     5401 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     5461 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     5521 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     5581 atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     5641 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     5701 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     5761 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     5821 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     5881 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     5941 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     6001 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     6061 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     6121 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     6181 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     6241 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     6301 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     6361 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     6421 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     6481 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     6541 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     6601 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     6661 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     6721 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     6781 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     6841 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     6901 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     6961 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     7021 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     7081 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     7141 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     7201 caagctcatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     7261 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     7321 cccgtggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     7381 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
     7441 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgaca tgattacgaa
     7501 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     7561 tgtatcttat catgtctgga tcaactggat aactcaagct aaccaaaatc atcccaaact
     7621 tcccacccca taccctatta ccactgccaa ttacctgtgg tttcatttac tctaaacctg
     7681 tgattcctct gaattatttt cattttaaag aaattgtatt tgttaaatat gtactacaaa
     7741 cttagtagt
//
