LOCUS       sequence-234794-        6702 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of GFP tagged CD274.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6702)
  AUTHORS   Ma W, Mayr C
  TITLE     A Membraneless Organelle Associated with the Endoplasmic Reticulum 
            Enables 3'UTR-Mediated Protein-Protein Interactions.
  JOURNAL   Cell. 2018 Oct 30. pii: S0092-8674(18)31315-1. doi: 
            10.1016/j.cell.2018.10.007.
  PUBMED    30449617
REFERENCE   2  (bases 1 to 6702)
  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: "Cell. 2018 
            Oct 30. pii: S0092-8674(18)31315-1. doi: 
            10.1016/j.cell.2018.10.007."
COMMENT     Sequence Label: SP-GFP-CD274-NU
FEATURES             Location/Qualifiers
     source          1..6702
                     /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..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(961..963,964..966,967..1677)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1006..1027)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1267..1286)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1614..1635
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2542..2559)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2548..2772
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2818..3246
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2905..2924)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3115..3136
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3255..3275)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3260..3589
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3440..3575
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3502..3521
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3602..4201
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3602..3621)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4098..4118
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4375..4496
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4412..4431)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4466..4485
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4545..4561)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4545..4561)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4558..4580)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4569..4585
                     /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(4593..4599,4600..4617,4618..4623))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4638..4659
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4776..4793)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4947..5535)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5027..5046)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5706..6497,6498..6566))
                     /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     6329..6348
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6567..6671)
                     /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 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gccaccatga ggatatttgc tgtctttata ttcatgacct actggcattt gctgaacgca
      961 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     1021 ggcgacgtaa acggccacaa gttcagcgtg tctggcgagg gcgagggcga tgccacctac
     1081 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1141 ctcgtgacca cccttaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     1201 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     1261 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1321 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1381 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     1441 ggcatcaagg cgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     1501 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     1561 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     1621 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagttt
     1681 actgtcacgg ttcccaagga cctatatgtg gtagagtatg gtagcaatat gacaattgaa
     1741 tgcaaattcc cagtagaaaa acaattagac ctggctgcac taattgtcta ttgggaaatg
     1801 gaggataaga acattattca atttgtgcat ggagaggaag acctgaaggt tcagcatagt
     1861 agctacagac agagggcccg gctgttgaag gaccagctct ccctgggaaa tgctgcactt
     1921 cagatcacag atgtgaaatt gcaggatgca ggggtgtacc gctgcatgat cagctatggt
     1981 ggtgccgact acaagcgaat tactgtgaaa gtcaatgccc catacaacaa aatcaaccaa
     2041 agaattttgg ttgtggatcc agtcacctct gaacatgaac tgacatgtca ggctgagggc
     2101 taccccaagg ccgaagtcat ctggacaagc agtgaccatc aagtcctgag tggtaagacc
     2161 accaccacca attccaagag agaggagaag cttttcaatg tgaccagcac actgagaatc
     2221 aacacaacaa ctaatgagat tttctactgc acttttagga gattagatcc tgaggaaaac
     2281 catacagctg aattggtcat cccagaacta cctctggcac atcctccaaa tgaaaggact
     2341 cacttggtaa ttctgggagc catcttatta tgccttggtg tagcactgac attcatcttc
     2401 cgtttaagaa aagggagaat gatggatgtg aaaaaatgtg gcatccaaga tacaaactca
     2461 aagaagcaaa gtgatacaca tttggaggag acgtaaggcg gccgctcgag tctagagggc
     2521 ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     2581 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     2641 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     2701 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     2761 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg
     2821 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2881 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     2941 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     3001 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     3061 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     3121 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     3181 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     3241 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     3301 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     3361 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     3421 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     3481 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
     3541 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccaac
     3601 catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt
     3661 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga
     3721 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
     3781 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga
     3841 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
     3901 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
     3961 gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg
     4021 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
     4081 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
     4141 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg
     4201 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     4261 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4321 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     4381 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4441 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4501 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     4561 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4621 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4681 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4741 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4801 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4861 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4921 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4981 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5041 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5101 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5161 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5221 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5281 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5341 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5401 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5461 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5521 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5581 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5641 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5701 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5761 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5821 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5881 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5941 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6001 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6061 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6121 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6181 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6241 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6301 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6361 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6421 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6481 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6541 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6601 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6661 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
