LOCUS       sequence-245743-        7732 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Constitutive, ubiquitous blue fluorescence protein (BFP) expression.
            .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7732)
  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 7732)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-TagBFP-W
FEATURES             Location/Qualifiers
     source          1..7732
                     /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"
     CDS             3412..4113
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    4122..4710
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4175..4195)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4593..4604)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4785..5018
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(5137..5155)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5223..5327
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5328..5396,5397..6188)
                     /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(5546..5565)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6359..6947
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6848..6867
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7101..7118
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7358..7379
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7394..7399,7400..7417,7418..7424)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7432..7448
                     /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     7437..7459
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7456..7472
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7456..7472
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7522..7541
                     /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 catgagcgag
     3421 ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgga caaccatcac
     3481 ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac catgagaatc
     3541 aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac tagcttcctc
     3601 tacggcagca agaccttcat caaccacacc cagggcatcc ccgacttctt caagcagtcc
     3661 ttccctgagg gcttcacatg ggagagagtc accacatacg aagacggggg cgtgctgacc
     3721 gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa gatcagaggg
     3781 gtgaacttca catccaacgg ccctgtgatg cagaagaaaa cactcggctg ggaggccttc
     3841 accgagacgc tgtaccccgc tgacggcggc ctggaaggca gaaacgacat ggccctgaag
     3901 ctcgtgggcg ggagccatct gatcgcaaac atcaagacca catatagatc caagaaaccc
     3961 gctaagaacc tcaagatgcc tggcgtctac tatgtggact acagactgga aagaatcaag
     4021 gaggccaaca acgagaccta cgtcgagcag cacgaggtgg cagtggccag atactgcgac
     4081 ctccctagca aactggggca caagcttaat taaaggatcc taatcaacct ctggattaca
     4141 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     4201 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     4261 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     4321 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     4381 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     4441 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     4501 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga
     4561 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     4621 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     4681 gtcggatctc cctttgggcc gcctccccgc ctgagatcct ttaagaccaa tgacttacaa
     4741 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca
     4801 ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat
     4861 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     4921 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     4981 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat gtcatcttat
     5041 tattcagtat ttataacttg caaagaaatg aatatcagag agtgagaggc ccgggttaat
     5101 taaggaaagg gctagatcat tcttgaagac gaaagggcct cgtgatacgc ctatttttat
     5161 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     5221 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5281 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     5341 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5401 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     5461 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5521 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg
     5581 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5641 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5701 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     5761 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     5821 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     5881 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     5941 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     6001 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     6061 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     6121 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6181 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     6241 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     6301 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     6361 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6421 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6481 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     6541 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     6601 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6661 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6721 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6781 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     6841 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     6901 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     6961 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     7021 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     7081 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     7141 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagcaagctc atggctgact
     7201 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     7261 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctccccgtgg cacgacaggt
     7321 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
     7381 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     7441 gataacaatt tcacacagga aacagctatg acatgattac gaatttcaca aataaagcat
     7501 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     7561 ggatcaactg gataactcaa gctaaccaaa atcatcccaa acttcccacc ccatacccta
     7621 ttaccactgc caattacctg tggtttcatt tactctaaac ctgtgattcc tctgaattat
     7681 tttcatttta aagaaattgt atttgttaaa tatgtactac aaacttagta gt
//
