LOCUS       sequence-218422-        7993 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses CD28-PafA in mammalian cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7993)
  AUTHORS   Liu Q, Zheng J, Sun W, Huo Y, Zhang L, Hao P, Wang H, Zhuang M
  TITLE     A proximity-tagging system to identify membrane protein-protein 
            interactions.
  JOURNAL   Nat Methods. 2018 Aug 13. pii: 10.1038/s41592-018-0100-5. doi: 
            10.1038/s41592-018-0100-5.
  PUBMED    30104635
REFERENCE   2  (bases 1 to 7993)
  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: "Nat 
            Methods. 2018 Aug 13. pii: 10.1038/s41592-018-0100-5. doi: 
            10.1038/s41592-018-0100-5."
COMMENT     Sequence Label: pEF6a-CD28-PafA
FEATURES             Location/Qualifiers
     source          1..7993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..1179
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          232..1170
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1127..1147
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3429..3458
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3525..3554
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3570..3587
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3610..3627)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3616..3840
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3886..4314
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3973..3992)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4183..4204
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4323..4343)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4328..4657
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4508..4643
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4570..4589
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4705..4752
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4771..5169
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5327..5448
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5364..5383)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5418..5437
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5497..5513)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5497..5513)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5510..5532)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5521..5537
                     /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(5545..5551,5552..5569,5570..5575))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5590..5611
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5728..5745)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5888..6476)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5968..5987)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6647..7438,7439..7507))
                     /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     7270..7289
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7508..7612)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7687..7706)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
       61 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
      121 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
      181 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
      241 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
      301 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      361 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      421 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      481 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      541 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      601 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      661 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      721 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
      781 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
      841 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
      901 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
      961 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1021 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1081 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1141 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gctagcgaat tctcacgcgt
     1201 caagtggagc aaggcaggtg gacagtggat ccttgacttg cggcgccacc atgctcaggc
     1261 tgctcttggc tctcaactta ttcccttcaa ttcaagtaac aggaaacaag attttggtga
     1321 agcagtcgcc catgcttgta gcgtacgaca atgcggtcaa ccttagctgc aagtattcct
     1381 acaatctctt ctcaagggag ttccgggcat cccttcacaa aggactggat agtgctgtgg
     1441 aagtctgtgt tgtatatggg aattactccc agcagcttca ggtttactca aaaacggggt
     1501 tcaactgtga tgggaaattg ggcaatgaat cagtgacatt ctacctccag aatttgtatg
     1561 ttaaccaaac agatatttac ttctgcaaaa ttgaagttat gtatcctcct ccttacctag
     1621 acaatgagaa gagcaatgga accattatcc atgtgaaagg gaaacacctt tgtccaagtc
     1681 ccctatttcc cggaccttct aagccctttt gggtgctggt ggtggttggt ggagtcctgg
     1741 cttgctatag cttgctagta acagtggcct ttattatttt ctgggtgagg agtaagagga
     1801 gcaggctcct gcacagtgac tacatgaaca tgactccccg ccgccccggg cccacccgca
     1861 agcattacca gccctatgcc ccaccacgcg acttcgcagc ctatcgctcc tctagagggc
     1921 ccttcggctc cggtggcggc agcggtgggg gcagtggagg cggaagcgga gggggctctg
     1981 gcatgagtac cgtggaatcc gcattgaccc gcaggatcat gggcattgaa acggagtatg
     2041 gcctcacctt tgttgatggt gattccaaaa agcttcgccc agatgagata gctcgaagga
     2101 tgtttcgtcc catcgtggag aaatattcca gctctaatat cttcataccc aatggttccc
     2161 ggttgtatct tgatgtgggt tcccacccgg agtacgccac cgccgagtgt gataatttga
     2221 cccagctgat caattttgaa aaagctggcg atgttattgc agatcgcatg gctgtagatg
     2281 ccgaagagtc gctggcgaaa gaagacattg ctgggcaggt gtacctgttt aaaaacaatg
     2341 tcgattccgt gggcaattct tatggctgcc acgaaaacta ccttgtgggt cgctccatgc
     2401 cgttgaaggc gttgggtaaa aggctgatgc cgtttctgat tacccgccag ctcatctgcg
     2461 gcgccggcag gatccatcac cccaatcctt tggataaagg cgaatccttc cccttgggct
     2521 actgcatatc ccagcgctct gaccacgtgt gggagggcgt atcaagtgcc accactagat
     2581 cacgccccat tatcaacacc cgtgatgagc cacatgcgga ttcccattct taccgcaggc
     2641 tgcacgtgat tgtgggtgat gccaacatgg cagagcccag catcgcgttg aaggtcggct
     2701 ccacgttgct ggttctggaa atgattgagg cagatttcgg tttgcccagc ttagagcttg
     2761 ccaatgatat tgcctcaatt agggaaatct cccgcgatgc aacaggatcc acactgttgt
     2821 ccctgaaaga tggcaccacc atgactgcct tgcagatcca gcaggtggtc tttgagcatg
     2881 cctcgaagtg gttggagcag cgccccgaac cagaattttc tggcacctcc aacacagaga
     2941 tggcccgcgt gctggatctg tggggtcgca tgttgaaagc gattgagtcc ggtgatttca
     3001 gcgaagtgga tacagaaatt gactgggtga tcaaaaagaa gctcattgat cgtttcattc
     3061 agcgcggcaa ccttgggttg gatgatccaa aacttgccca agtggacttg acttatcacg
     3121 atattaggcc aggtagaggc ctatttagcg tgctgcaaag ccgcggcatg atcaaacggt
     3181 ggactactga tgaggcgatt ttagctgcgg tggataccgc tcctgataca acacgtgctc
     3241 atttgcgcgg gcgaatcctt aaagcggcgg atactctggg agtacctgtg actgtcgatt
     3301 ggatgcgtca caaggtcaac cgaccggagc cacaatcggt ggaattgggg gatccttttt
     3361 ccgcagtgaa ttccgaggtt gatcagttga ttgagtatat gaccgttcat gcggagagct
     3421 atcgcagtga acaaaaactc atctcagaag aggatctgaa tatgcagtaa gaattctgca
     3481 gatatccagc acagtggcgg ccgctcgagt ctagagggcc cttcgaacaa aaactcatct
     3541 cagaagagga tctgaatatg cataccggtc atcatcacca tcaccattga gtttaaaccc
     3601 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     3661 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     3721 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     3781 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     3841 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
     3901 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3961 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     4021 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     4081 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     4141 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     4201 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     4261 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
     4321 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     4381 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     4441 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     4501 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     4561 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
     4621 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
     4681 cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata
     4741 gtataatacg acaaggtgag gaactaaacc atggccaagc ctttgtctca agaagaatcc
     4801 accctcattg aaagagcaac ggctacaatc aacagcatcc ccatctctga agactacagc
     4861 gtcgccagcg cagctctctc tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat
     4921 tttactgggg gaccttgtgc agaactcgtg gtgctgggca ctgctgctgc tgcggcagct
     4981 ggcaacctga cttgtatcgt cgcgatcgga aatgagaaca ggggcatctt gagcccctgc
     5041 ggacggtgcc gacaggtgct tctcgatctg catcctggga tcaaagccat agtgaaggac
     5101 agtgatggac agccgacggc agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg
     5161 gagggctaag cacttcgtgg ccgaggagca ggactgacac gtgctacgag atttcgattc
     5221 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     5281 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     5341 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     5401 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     5461 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     5521 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     5581 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     5641 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5701 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     5761 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5821 ggataacgca ggaaagaaca tgtgagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5881 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5941 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     6001 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     6061 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     6121 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6181 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6241 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6301 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     6361 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6421 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6481 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6541 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6601 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6661 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6721 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6781 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6841 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6901 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6961 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     7021 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     7081 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     7141 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     7201 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7261 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7321 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7381 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7441 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7501 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7561 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7621 cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac
     7681 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
     7741 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
     7801 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
     7861 ggccagatat acgcgttgac attgattatt gactaggctt ttgcaaaaag ctttgcaaag
     7921 atggataaag ttttaaacag agaggaatct ttgcagctaa tggaccttct aggtcttgaa
     7981 aggagtggga att
//
