LOCUS       sequence-202211-        7387 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP-PLC pleckstrin homology domain linked to FAP for CALI
            validation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7387)
  AUTHORS   He J, Wang Y, Missinato MA, Onuoha E, Perkins LA, Watkins SC, St 
            Croix CM, Tsang M, Bruchez MP
  TITLE     A genetically targetable near-infrared photosensitizer.
  JOURNAL   Nat Methods. 2016 Mar;13(3):263-268. doi: 10.1038/nmeth.3735. Epub 
            2016 Jan 25.
  PUBMED    26808669
REFERENCE   2  (bases 1 to 7387)
  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/nmeth.3735"; journalName: "Nat Methods"; date: "2016-03"; 
            volume: "13"; issue: "3"; pages: "263-268"
COMMENT     Sequence Label: EGFP-PH-dL5**
FEATURES             Location/Qualifiers
     source          1..7387
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..616
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        617..820
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     771..791
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     865..884
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        865..883
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(919..921,922..924,925..1635)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(919..921,922..924,925..1635)
                     /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(964..985)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1225..1244)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1572..1593
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2203..2922
                     /codon_start=1
                     /product="fluorogen-activating protein composed of a dimer 
                     of the L5-MG single-chain antibody fragment (Szent-Gyorgyi 
                     et al., 2008)"
                     /label=FAP-alpha-2
                     /note="binds malachite green derivatives to produce red 
                     fluorescence"
                     /translation="QAVVTQEPSVTVSPGGTVILTCGSGTGAVTSGHYANWFQQKPGQA
                     PRALIFDTDKKYSWTPGRFSGSLLGAKAALTISDAQPEDEAEYYCSLSDVDGYLFGGGT
                     QLTVLSGGGGSGGGGSGGGGSGGGGSQAVVTQEPSVTVSPGGTVILTCGSGTGAVTSGH
                     YANWFQQKPGQAPRALIFDTDKKYSWTPGRFSGSLLGAKAALTISDAQPEDEAEYYCSL
                     SDVDGYLFGGGTQLTVLS"
     primer_bind     complement(2978..2995)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2984..3208
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3254..3682
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3341..3360)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3551..3572
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3691..3711)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3696..4025
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3876..4011
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3938..3957
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4092..4886
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4146..4165)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4756..4775
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5060..5181
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5097..5116)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5151..5170
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5230..5246)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5230..5246)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5243..5265)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5254..5270
                     /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(5278..5284,5285..5302,5303..5308))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5323..5344
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5461..5478)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5632..6220)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5712..5731)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6391..7182,7183..7251))
                     /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     7014..7033
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7252..7356)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      781 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc
      841 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta
      901 gcgtttaaac ttaagcttat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
      961 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     1021 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     1081 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     1141 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     1201 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     1261 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1321 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     1381 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     1441 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1501 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     1561 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     1621 gacgagctgt acaagggatc ccttgaattc atggactcgg gccgggactt cctgaccctg
     1681 cacggcctac aggatgatga ggatctacag gcgctgctga agggcagcca gctcctgaag
     1741 gtgaagtcca gctcatggag gagagagcgc ttctacaagt tgcaggagga ctgcaagacc
     1801 atctggcagg agtcccgcaa ggtcatgcgg accccggagt cccagctgtt ctccatcgag
     1861 gacattcagg aggtgcgaat ggggcaccgc acggagggtc tggagaagtt cgcccgtgat
     1921 gtgcccgagg accgctgctt ctccattgtc ttcaaggacc agcgcaatac actagacctc
     1981 atcgccccat cgccagctga tgcccagcac tgggtgctgg ggctgcacaa gatcatccac
     2041 cactcaggct ccatggacca gcgtcagaag ctacagcact ggattcactc ctgcttgcga
     2101 aaagctgaca aaaacaagga caacaagatg agcttcaagg agctgcagaa cttcctgaag
     2161 catatgggag gcggtgggtc tggtggcggt ggcagcaccg gtcaggccgt cgttacccaa
     2221 gaacctagtg ttaccgttag cccaggtggt actgttatac ttacttgtgg aagtggtacg
     2281 ggtgccgtca catctggtca ttatgcaaat tggtttcaac aaaaaccagg acaagctcca
     2341 agagctttga tttttgatac tgataagaag tattcttgga ccccaggtag attttctgga
     2401 tctttgctgg gagcaaaggc agctttgaca atatcagatg ctcagcctga ggacgaagcc
     2461 gagtattact gttctcttag cgacgtggat ggctacttgt ttggcggtgg aacacaactg
     2521 acggttctgt ccggtggtgg cggctctggt ggcggtggca gcggcggtgg tggttccgga
     2581 ggcggcggtt ctcaggctgt ggtgactcag gagccgtcag tgactgtgtc cccaggaggg
     2641 acagtcattc tcacttgtgg ctccggcact ggagctgtca ccagtggtca ttatgccaac
     2701 tggttccagc agaagcctgg ccaagccccc agggcactta tatttgacac cgacaagaag
     2761 tattcctgga cccctggccg attctcaggc tccctccttg gggccaaggc tgccctgacc
     2821 atctcggatg cgcagcctga agatgaggct gagtattact gttcgctctc cgacgttgac
     2881 ggttatctgt tcggaggagg cacccagctg accgtcctct cctaatagct cgaggcagct
     2941 gtcgactcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     3001 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     3061 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     3121 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     3181 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     3241 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3301 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3361 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3421 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3481 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3541 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3601 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3661 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3721 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3781 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3841 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3901 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3961 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     4021 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     4081 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     4141 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     4201 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     4261 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     4321 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     4381 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     4441 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     4501 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     4561 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     4621 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     4681 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     4741 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     4801 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     4861 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     4921 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     4981 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     5041 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     5101 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     5161 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     5221 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     5281 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     5341 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5401 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     5461 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5521 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5581 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5641 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5701 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5761 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5821 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5881 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5941 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     6001 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     6061 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     6121 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     6181 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     6241 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     6301 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6361 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6421 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6481 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6541 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6601 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6661 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6721 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6781 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6841 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6901 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6961 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     7021 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     7081 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     7141 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     7201 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     7261 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     7321 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     7381 tgacgtc
//
