LOCUS       sequence-316129-        7336 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression in mammalian cells of Phosphatidic Acid preferring 
            Phospholipase A1 tagged with EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7336)
  AUTHORS   Gormal RS, Padmanabhan P, Kasula R, Bademosi AT, Coakley S, 
            Giacomotto J, Blum A, Joensuu M, Wallis TP, Lo HP, Budnar S, Rae J, 
            Ferguson C, Bastiani M, Thomas WG, Pardon E, Steyaert J, Yap AS, 
            Goodhill GJ, Hilliard MA, Parton RG, Meunier FA
  TITLE     Modular transient nanoclustering of activated beta2-adrenergic 
            receptors revealed by single-molecule tracking of 
            conformation-specific nanobodies.
  JOURNAL   Proc Natl Acad Sci U S A. 2020 Nov 19. pii: 2007443117. doi: 
            10.1073/pnas.2007443117.
  PUBMED    33214152
REFERENCE   2  (bases 1 to 7336)
  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: "Proc Natl 
            Acad Sci U S A. 2020 Nov 19. pii: 2007443117. doi: 
            10.1073/pnas.2007443117."
COMMENT     Sequence Label: pEGFP-C1 PA-PLA1 (EGFP-PA-PLA1)
FEATURES             Location/Qualifiers
     source          1..7336
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(613..615,616..618,619..1329)
                     /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(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4124..4245
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4161..4180)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4215..4234
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4252..4707)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4389..4410)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4601..4620
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4734..4838
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4840..5197
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4840..5031
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4863..4883)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5048..5183
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5232..6026
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             5232..6026
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5286..5305)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5896..5915
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6214..6233)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6258..6305
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6634..7222
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7123..7142
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atctatgaat tacccgggcc gcgggtcccc acggagcccc
     1381 gagcataacg gccgaggcgg cggcggcggc gcctgggagc tgggctcaga cgcgaggcca
     1441 gcgttcggcg gcggcgtctg ctgcttcgag cacctgcccg gcggggaccc ggacgacggc
     1501 gacgtgcccc tggccctgct gcgcggggaa cccgggctgc atttggcgcc gggcaccgac
     1561 gaccacaacc accacctcgc gctggacccc tgcctcagtg acgagaacta tgacttcagc
     1621 tccgccgagt cgggctcctc gctgcgctac tacagcgagg gtgagagcgg cggcggcggc
     1681 agctccttgt cgctgcaccc gccgcagcag cctccgctgg tcccgacgaa ctcggggggc
     1741 ggcggcgcga caggagggtc ccccggggaa aggaaacgta cccggcttgg cggcccggcg
     1801 gcccggcacc gctatgaggt agtgacggag ctgggcccgg aggaggtacg ctggttctac
     1861 aaggaggaca agaagacctg gaagcccttc atcggctacg actcgctccg catcgagctc
     1921 gccttccgga ccctgctgca gaccacgggt gcccggcccc agggcgggga ccgggacggc
     1981 gaccatgtgt gctcccccac gggcccagcc tccagttccg gagaagatga cgatgaggac
     2041 cgcgcctgcg gcttctgcca gagtacgacg gggcacgagc cggagatggt ggagcttgtg
     2101 aacatcgagc ctgtgtgcgt gcggggcggc ctctacgagg tggatgtgac ccaaggagag
     2161 tgctacccgg tgtactggaa ccaggctgat aaaataccag taatgcgtgg acagtggttt
     2221 attgacggca cttggcagcc tctagaagag gaagaaagta atttaattga gcaagaacat
     2281 ctcaattgtt ttaggggcca gcagatgcag gaaaatttcg atattgaagt gtcaaaatcc
     2341 atagatggaa aagatgctgt tcatagtttc aagttgagtc gaaaccatgt ggactggcac
     2401 agtgtggatg aagtatatct ttatagtgat gcaacaacat ctaaaattgc aagaacagtt
     2461 acccaaaaac tgggattttc taaagcatca agtagtggta ccagacttca tagaggttat
     2521 gtagaagaag ccacattaga agacaagcca tcacagacta cccatattgt atttgttgtg
     2581 catggcattg ggcagaaaat ggaccaagga agaattatca aaaatacagc tatgatgaga
     2641 gaagctgcaa gaaaaataga agaaaggcat ttttccaacc atgcaacaca tgttgaattt
     2701 ctgcctgttg agtggcggtc aaaacttact cttgatggag acactgttga ttccattact
     2761 cctgacaaag tacgaggttt aagggatatg ctgaacagca gtgcaatgga cataatgtat
     2821 tatactagtc cactttatag agatgaacta gttaaaggcc ttcagcaaga gctgaatcga
     2881 ttgtattccc ttttctgttc tcggaatcca gactttgaag aaaaaggggg taaagtctca
     2941 atagtatcac attccttggg atgtgtaatt acttatgaca taatgactgg ctggaatcca
     3001 gttcggctgt atgaacagtt gctgcaaaag gaagaagagt tgcctgatga acgatggatg
     3061 agctatgaag aacgacatct tcttgatgaa ctctatataa caaaacgacg gctgaaggaa
     3121 atagaagaac ggcttcacgg attgaaagca tcatctatga cacaaacacc tgccttaaaa
     3181 tttaaggttg agaatttctt ctgtatggga tccccattag cagttttctt ggcgttgcgt
     3241 ggcatccgcc caggaaatac tggaagtcaa gaccatattt tgcctagaga gatttgtaac
     3301 cggttactaa atatttttca tcctacagat ccagtggctt atagattaga accattaata
     3361 ctgaaacact acagcaacat ttcacctgtc cagatccact ggtacaatac ttcaaatcct
     3421 ttaccttatg aacatatgaa gccaagcttt ctcaacccag ctaaagaacc tacctcagtt
     3481 tcagagaatg aaggcatttc aaccatacca agccctgtga cctcaccagt tttgtcccgc
     3541 cgacactatg gagaatctat aacaaatata ggcaaagcaa gcatattagg ggctgctagc
     3601 attggaaagg gacttggagg aatgttgttc tcaagatttg gacgttcatc tacaacacag
     3661 tcatctgaaa catcaaaaga ctcaatggaa gatgagaaga agccagttgc ctcaccttct
     3721 gctaccaccg tagggacaca gacccttcca catagcagtt ctggcttcct cgattctgca
     3781 ttggagttgg atcacaggat tgattttgaa ctcagagaag gccttgtgga gagccgctat
     3841 tggtcagctg tcacgtcgca tactgcctat tggtcatcct tggatgttgc cctttttctt
     3901 ttaaccttca tgtataaaca tgagcacgat gatgatgcaa aacccaattt agatccaatc
     3961 tgagaattct gcagtcgacg gtaccgcggg cccgggatcc accggatcta gataactgat
     4021 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
     4081 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     4141 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     4201 actgcattct agttgtggtt tgtccaaact catcaatgta tcttaacgcg taaattgtaa
     4261 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     4321 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     4381 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     4441 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     4501 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     4561 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     4621 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     4681 cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa
     4741 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4801 cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct
     4861 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     4921 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     4981 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     5041 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     5101 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     5161 gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag
     5221 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     5281 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     5341 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     5401 tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     5461 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     5521 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     5581 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     5641 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     5701 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga
     5761 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     5821 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     5881 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     5941 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     6001 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     6061 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     6121 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     6181 ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga
     6241 aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt
     6301 tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc
     6361 ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg
     6421 tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta
     6481 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     6541 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     6601 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     6661 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6721 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6781 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     6841 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6901 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6961 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     7021 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     7081 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     7141 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     7201 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     7261 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     7321 tattaccgcc atgcat
//
