LOCUS       sequence-405847-        4529 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  knock-in donor vector of GFP to the C-terminus of endogenous human 
            PLIN2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4529)
  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 4529)
  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
FEATURES             Location/Qualifiers
     source          1..4529
                     /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"
     primer_bind     complement(2532..2549)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2532..2548)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2541..2563)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2757..2776)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2876..2898
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2936..2954)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3022..3126
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3127..3195,3196..3987)
                     /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(3345..3364)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4158..4529,1..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 ttacaaatag taaacctggc
     1921 cgtatcacta gtgcatgctg tggccagaca gatgacacct tttgttatgt tgaaattaac
     1981 ttgctaggca accctaaatt gggaagcaag tagctagtat aaaggccctc aattgtagtt
     2041 gtttccagct gaattaagag ctttaaagtt tctggcatta gcagatgatt tctgttcacc
     2101 tggtaagaaa agaatgatag gcttgtcaga gcctatagcc agaactcaga aaaaattcaa
     2161 atgcacttat gttctcattc tatggccatt gtgttgcctc tgttactgtt tgtattgaat
     2221 aaaaacatct tcatgtgggc tggggtagaa actggtgtct gctctggtgt gatctgaaag
     2281 gcgtcttcac tgctttatct catgatgctt gcttgtaaaa cttgatttta gtttttcatt
     2341 tctcaaatag gaatactacc tttgaattca ataaaattca ctgcaggata gaccagttac
     2401 atgctgtttg ttccatatgc tttgtgtgtt gctttcgtag agctgcttaa cctgcatgac
     2461 agagttatta tacatacatg aatttcaatt ggcctgttgg ggagggatcc ccgggtaccg
     2521 agctcgaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     2581 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     2641 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg
     2701 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca
     2761 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     2821 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     2881 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     2941 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     3001 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3061 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     3121 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     3181 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     3241 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     3301 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     3361 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     3421 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     3481 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     3541 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     3601 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     3661 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     3721 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     3781 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     3841 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     3901 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     3961 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     4021 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     4081 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4141 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4201 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4261 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     4321 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4381 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4441 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4501 agcttggagc gaacgaccta caccgaact
//
