LOCUS       sequence-407622-        5760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  knock-in donor vector of GFP-P2A-BFP to C-terminus of endogenous 
            human PLIN2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5760)
  AUTHORS   Roberts MA, Deol KK, Mathiowetz AJ, Lange M, Leto DE, Stevenson J, 
            Hashemi SH, Morgens DW, Easter E, Heydari K, Nalls MA, Bassik MC, 
            Kampmann M, Kopito RR, Faghri F, Olzmann JA
  TITLE     Parallel CRISPR-Cas9 screens identify mechanisms of PLIN2 and lipid 
            droplet regulation.
  JOURNAL   Dev Cell. 2023 Jul 18:S1534-5807(23)00331-3. doi: 
            10.1016/j.devcel.2023.07.001.
  PUBMED    37494933
REFERENCE   2  (bases 1 to 5760)
  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: "Dev Cell. 
            2023 Jul 18:S1534-5807(23)00331-3. doi: 
            10.1016/j.devcel.2023.07.001."
COMMENT     Sequence Label: pUC19/PLIN2 GFP-P2A-BFP
FEATURES             Location/Qualifiers
     source          1..5760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     118..137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     371..388
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    505..526
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(541..546,547..564,565..571)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    579..595
                     /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     584..606
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     603..619
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     603..619
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(1191..1193,1194..1196,1197..1907)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             1917..1973
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(3294..3311)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3294..3310)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3303..3325)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4107..4129
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4167..4185)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4253..4357
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4358..4426,4427..5218)
                     /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     complement(4576..4595)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5389..5760,1..217)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
       61 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
      121 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
      181 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
      241 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
      301 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
      361 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
      421 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
      481 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
      541 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
      601 cacaggaaac agctatgacc atgattacgc caagcttgca tgcctgcagg tcgactctag
      661 agaggatttt taaaagccaa agaatccaca tatgtggtgg acacataacc agcctactgt
      721 catcgaacta atttatacat agcactgttg gatccactgc taattttaag ttttattttt
      781 agcacattga gtcacgtact cttgcaattg cccgcaacct gactcagcag ctccagacca
      841 cgtgccacac cctcctgtcc aacatccaag gtgtaccaca gaacatccaa gatcaagcca
      901 agcacatggg ggtgatggca ggcgacatct actcagtgtt ccgcaatgct gcctccttta
      961 aagaagtgtc tgacagcctc ctcacttcta gcaaggggca gctgcagaaa atgaaggaat
     1021 ctttagatga cgtgatggat tatcttgtta acaacacgcc cctcaactgg ctggtaggtc
     1081 ccttttatcc tcagctgact gagtctcaga atgctcagga ccaaggtgca gagatggaca
     1141 agagcagcca ggagacccag cgatctgagc ataaaactca tggcagcggc atggtcagca
     1201 agggggaaga gctgttcact ggtgtagtcc ccatacttgt agagcttgac ggtgacgtga
     1261 acggacataa atttagcgtt tcaggcgagg gagagggtga cgcgacatat gggaaactga
     1321 cactcaagtt catatgtact actggtaagc tccctgtgcc ctggccgact ctggttacca
     1381 cgcttactta tggtgtacaa tgtttcagta gatatccgga ccatatgaag caacatgact
     1441 tctttaaaag tgccatgcct gaagggtatg tacaagaacg cacaattttt tttaaagacg
     1501 atggtaatta taagactagg gcggaggtta aattcgaggg agatacgctg gtaaaccgca
     1561 ttgagcttaa gggcattgat ttcaaagagg acggcaacat tttgggacac aaacttgaat
     1621 ataactacaa tagtcataac gtttacatta tggcggacaa acaaaaaaac gggataaaag
     1681 tgaactttaa gattcgccat aatatagagg atggaagtgt acaactggcg gatcactacc
     1741 agcagaacac tcccattggt gacggtcccg tcttgctgcc cgataatcac tacttgtcca
     1801 cgcagagtgc gttgagtaag gatccgaatg aaaagaggga ccatatggtg ctccttgaat
     1861 ttgttaccgc agcgggcatc acgctcggca tggacgagct ttacaaaggc agcggtgcta
     1921 caaacttctc cctcctcaag caagctgggg atgtggaaga aaaccctgga cccagcgagt
     1981 tgatcaagga aaatatgcat atgaaactgt atatggaggg aactgtagac aatcaccact
     2041 tcaagtgcac gtctgaaggg gaagggaagc cttatgaggg gactcagact atgaggataa
     2101 aagtagtaga ggggggtcca ctccccttcg ccttcgatat actcgctacg tcattcctct
     2161 atggctcaaa aacttttata aatcatacac agggtattcc tgatttcttc aaacagtctt
     2221 ttcctgaagg attcacctgg gagagagtga caacctacga agacgggggc gtgcttacgg
     2281 ccacgcaaga tacatccctt caggatggct gccttattta taatgtgaaa attcgcgggg
     2341 ttaactttac aagcaacggg ccagtgatgc agaagaagac attgggctgg gaagccttta
     2401 ccgaaacact gtaccctgca gacggcggcc tggaaggaag gaacgatatg gcccttaagt
     2461 tggtgggcgg tagccacctt atcgctaata tcaaaacaac ctaccgcagt aaaaagccag
     2521 ctaaaaatct taagatgcca ggggtgtatt atgttgatta tagactggaa cgcataaaag
     2581 aggccaataa tgaaacatac gtcgaacagc atgaggtagc cgtagcccgg tactgtgatc
     2641 tccccagcaa gttggggcat aaacttaact agtaaacctg gccgtatcac tagtgcatgc
     2701 tgtggccaga cagatgacac cttttgttat gttgaaatta acttgctagg caaccctaaa
     2761 ttgggaagca agtagctagt ataaaggccc tcaattgtag ttgtttccag ctgaattaag
     2821 agctttaaag tttctggcat tagcagatga tttctgttca cctggtaaga aaagaatgat
     2881 aggcttgtca gagcctatag ccagaactca gaaaaaattc aaatgcactt atgttctcat
     2941 tctatggcca ttgtgttgcc tctgttactg tttgtattga ataaaaacat cttcatgtgg
     3001 gctggggtag aaactggtgt ctgctctggt gtgatctgaa aggcgtcttc actgctttat
     3061 ctcatgatgc ttgcttgtaa aacttgattt tagtttttca tttctcaaat aggaatacta
     3121 cctttgaatt caataaaatt cactgcagga tagaccagtt acatgctgtt tgttccatat
     3181 gctttgtgtg ttgctttcgt agagctgctt aacctgcatg acagagttat tatacataca
     3241 tgaatttcaa ttggcctgtt ggggagggat ccccgggtac cgagctcgaa ttcactggcc
     3301 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     3361 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     3421 caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat
     3481 ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca
     3541 tagttaagcc agccccgaca cccgccaaca cccgctgact agggcggagg ttaaattcga
     3601 gggagatacg ctggtaaacc gcattgagct taagggcatt gatttcaaag aggacggcaa
     3661 cattttggga cacaaacttg aatataacta caatagtcat aacgtttaca ttatggcgga
     3721 caaacaaaaa aacgggataa aagtgaactt taagattcgc cataatatag aggatggaag
     3781 tgtacaactg gcggatcact accagcagaa cactcccatt ggtgacggtc ccgtcttgct
     3841 gcccgataat cactacttgt ccacgcagag tgcgttgagt aaggatccga atgaaaagag
     3901 ggaccatatg gtgctccttg aatttgttac cgcagcgggc atcacgctcg gcatggacga
     3961 gctttacaaa atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     4021 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     4081 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4141 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     4201 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     4261 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     4321 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     4381 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     4441 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     4501 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     4561 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     4621 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     4681 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     4741 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     4801 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     4861 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     4921 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     4981 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     5041 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     5101 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     5161 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     5221 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     5281 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     5341 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     5401 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     5461 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     5521 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     5581 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     5641 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     5701 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
//
