LOCUS       sequence-237908-        8364 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector expressing EX-HA-FRB-Cb5 on mammalian ER membrane.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8364)
  AUTHORS   Han Y, Branon TC, Martell JD, Boassa D, Shechner D, Ellisman MH, 
            Ting A
  TITLE     Directed Evolution of Split APEX2 Peroxidase.
  JOURNAL   ACS Chem Biol. 2019 Mar 8. doi: 10.1021/acschembio.8b00919.
  PUBMED    30848125
REFERENCE   2  (bases 1 to 8364)
  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: "ACS Chem 
            Biol. 2019 Mar 8. doi: 10.1021/acschembio.8b00919."
COMMENT     Sequence Label: EX-HA-FRB-Cb5_pLX304
FEATURES             Location/Qualifiers
     source          1..8364
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        65..368
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        369..572
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     523..543
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    595..619
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    1371..1959
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1424..1444)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1842..1853)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2031..2264
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2342..2463
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2379..2398)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2433..2452
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2503..2638
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2658..2677)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2659..2677)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2687..2704)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2687..2703)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2696..2718)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2845..3300
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2932..2951)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3142..3163
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3326..3430
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3431..3499,3500..4291)
                     /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(3649..3668)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4462..5050
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4951..4970
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5204..5221
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5338..5359
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5374..5379,5380..5397,5398..5404)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5412..5428
                     /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     5417..5439
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5436..5452
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5436..5452
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5471..5491
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5473..5491
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5519..5745
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5746..5926
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5973..6098
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6591..6824
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7009..7053
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        7228..7728
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     7647..7665
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             7740..8135
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    8195..8312
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 cccggggtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
       61 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
      121 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      181 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      241 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
      301 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      361 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
      421 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
      481 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
      541 gtgtacggtg ggaggtctat ataagcagag ctctctggct aactgtcggg atcaacaagt
      601 ttgtacaaaa aagcaggctt cgaaggagat agaaccatgg ggctgctgca gctgcccagc
      661 gacaaagccc tgctgtccga tcccgtgttc agacctctgg tcgataagta tgcagccgac
      721 gaggatgctt ttttcgcaga ttacgcagaa gcacatcaga agctgtcaga actgggattt
      781 gccgacgccg gctcgggcgg gagtaccggt tacccatacg acgttccaga ctacggtagt
      841 gggagcggtg tggccatcct gtggcacgag atgtggcatg aaggcctgga agaggcatct
      901 cgtttgtact ttggggaaag gaacgtgaaa ggcatgtttg aggtgctgga gcccttgcat
      961 gctatgatgg aacggggccc ccagactctg aaggaaacat cctttaatca ggcctatggt
     1021 cgagatttaa tggaggccca agagtggtgc aggaagtaca tgaaatcagg gaatgtcaag
     1081 gacctcaccc aagcctggga cctctattat catgtgttcc gacgaatctc aagtgctggt
     1141 ggtagtgctg gtggtagtgc tggtggtagt gctggtggta gtgctggtgg tcctcgagct
     1201 caagcttcga attcgagtgc tggtggtatc accaccgtgg agtccaactc ctcctggtgg
     1261 accaactggg tgatccccgc catctccgcc ctggtggtgg ccctgatgta ccgcctgtac
     1321 atggccgagg actagtaagc tagcgctaac cggtggcgcg ttaagtcgac aatcaacctc
     1381 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     1441 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     1501 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     1561 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     1621 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     1681 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     1741 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
     1801 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     1861 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     1921 ctcagacgag tcggatctcc ctttgggccg cctccccgcg tcgactttaa gaccaatgac
     1981 ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct
     2041 aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga
     2101 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     2161 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     2221 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtacgt atagtagttc
     2281 atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag
     2341 gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     2401 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     2461 ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccatcccgcc cctaactccg
     2521 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     2581 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     2641 gggacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
     2701 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     2761 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     2821 tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2881 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2941 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3001 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     3061 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3121 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     3181 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3241 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     3301 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3361 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3421 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3481 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3541 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     3601 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     3661 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3721 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3781 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3841 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3901 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3961 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     4021 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     4081 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     4141 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     4201 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     4261 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     4321 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4381 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4441 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4501 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4561 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     4621 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4681 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4741 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4801 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4861 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4921 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4981 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     5041 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     5101 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     5161 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     5221 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     5281 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     5341 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     5401 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     5461 cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc aagcttaatg
     5521 tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc
     5581 ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt
     5641 gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac tgaattgccg
     5701 cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc tctctggtta
     5761 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     5821 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     5881 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca
     5941 gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg
     6001 aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta
     6061 gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta
     6121 gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac
     6181 atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa
     6241 catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag
     6301 aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag
     6361 agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga
     6421 ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat
     6481 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     6541 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     6601 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     6661 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     6721 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     6781 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     6841 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     6901 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     6961 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     7021 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     7081 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     7141 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     7201 acccacctcc caaccccgag gggacccttg cgccttttcc aaggcagccc tgggtttgcg
     7261 cagggacgcg gctgctctgg gcgtggttcc gggaaacgca gcggcgccga ccctgggtct
     7321 cgcacattct tcacgtccgt tcgcagcgtc acccggatct tcgccgctac ccttgtgggc
     7381 cccccggcga cgcttcctgc tccgccccta agtcgggaag gttccttgcg gttcgcggcg
     7441 tgccggacgt gacaaacgga agccgcacgt ctcactagta ccctcgcaga cggacagcgc
     7501 cagggagcaa tggcagcgcg ccgaccgcga tgggctgtgg ccaatagcgg ctgctcagca
     7561 gggcgcgccg agagcagcgg ccgggaaggg gcggtgcggg aggcggggtg tggggcggta
     7621 gtgtgggccc tgttcctgcc cgcgcggtgt tccgcattct gcaagcctcc ggagcgcacg
     7681 tcggcagtcg gctccctcgt tgaccgaatc accgacctct ctccccaggg ggtaccacca
     7741 tggccaagcc tttgtctcaa gaagaatcca ccctcattga aagagcaacg gctacaatca
     7801 acagcatccc catctctgaa gactacagcg tcgccagcgc agctctctct agcgacggcc
     7861 gcatcttcac tggtgtcaat gtatatcatt ttactggggg accttgtgca gaactcgtgg
     7921 tgctgggcac tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc gcgatcggaa
     7981 atgagaacag gggcatcttg agcccctgcg gacggtgccg acaggtgctt ctcgatctgc
     8041 atcctgggat caaagccata gtgaaggaca gtgatggaca gccgacggca gttgggattc
     8101 gtgaattgct gccctctggt tatgtgtggg agggcctgca gctgcagtag taagaattct
     8161 agatcttgag acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg
     8221 ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat
     8281 tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc
     8341 cactttggcg ccggctcgag gggg
//
