LOCUS       sequence-296745-        9048 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpression of HLA-E fused to B2M.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9048)
  TITLE     Lian Lab CRISPR plasmids
REFERENCE   2  (bases 1 to 9048)
  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"
COMMENT     Sequence Label: pLR02_pXL001_HLAEB2M
FEATURES             Location/Qualifiers
     source          1..9048
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             117..750
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    797..922
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1415..1648
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1833..1877
                     /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"
     misc_feature    2164..2281
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2410..3588
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2641..3579
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3536..3556
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     misc_feature    5275..5737
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5331..5348)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5558..5577
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5770..6369
                     /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(5770..5789)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6266..6286
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             6572..6805
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6956..6973)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6962..7186
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     regulatory      7229..7238
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        7282..7386
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7387..7455,7456..8247)
                     /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(7605..7624)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8418..9006
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8907..8926
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cggaccgcca ctgccaatta cctgtggttt catttactct aaacctgtga ttcctctgaa
       61 ttattttcat tttaaagaaa ttgtatttgt taaatatgta ctacaaactt agtagttgga
      121 agggctaatt cactcccaaa gaagacaaga tatccttgat ctgtggatct accacacaca
      181 aggctacttc cctgattagc agaactacac accagggcca ggggtcagat atccactgac
      241 ctttggatgg tgctacaagc tagtaccagt tgagccagat aaggtagaag aggccaataa
      301 aggagagaac accagcttgt tacaccctgt gagcctgcat gggatggatg acccggagag
      361 agaagtgtta gagtggaggt ttgacagccg cctagcattt catcacgtgg cccgagagct
      421 gcatccggag tacttcaaga actgctgata tcgagcttgc tacaagggac tttccgctgg
      481 ggactttcca gggaggcgtg gcctgggcgg gactggggag tggcgagccc tcagatcctg
      541 catataagca gctgcttttt gcctgtactg ggtctctctg gttagaccag atctgagcct
      601 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
      661 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
      721 ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa
      781 agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag
      841 aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga
      901 gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa
      961 aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag
     1021 cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag
     1081 acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt
     1141 atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa
     1201 ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc
     1261 ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat
     1321 ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa
     1381 gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg
     1441 gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat
     1501 tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc
     1561 atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg
     1621 aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt
     1681 gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga
     1741 atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact
     1801 ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag
     1861 ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat
     1921 tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta
     1981 tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc
     2041 cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca
     2101 gatccattcg attagtgaac ggatctcgac ggtatcgcca caaatggcag tattcatcca
     2161 caattttaaa agaaaggggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     2221 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
     2281 tcgggtttat tacagggaca gcagagatcc actttggatc gataagcttt gcaaagatgg
     2341 ataaagtttt aaacagagag gaatctttgc agctaatgga ccttctaggt cttgaaagga
     2401 gtgggaattg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag
     2461 aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac
     2521 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     2581 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     2641 gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg
     2701 ccttgaatta cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga
     2761 agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt
     2821 gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt
     2881 ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt
     2941 tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt
     3001 tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg
     3061 ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct
     3121 ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg
     3181 tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca
     3241 aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg
     3301 gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg
     3361 cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt
     3421 tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac
     3481 ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag
     3541 cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgagg aattgatcct
     3601 tcgaactagt atggtagatg gaaccctcct tttactcctc tcggaggccc tggcccttac
     3661 ccagacctgg gcgggctccc actccttgaa gtatttccac acttccgtgt cccggcccgg
     3721 ccgcggggag ccccgcttca tctctgtggg ctacgtggac gacacccagt tcgtgcgctt
     3781 cgacaacgac gccgcgagtc cgaggatggt gccgcgggcg ccgtggatgg agcaggaggg
     3841 gtcagagtat tgggaccggg agacacggag cgccagggac accgcacaga ttttccgagt
     3901 gaatctgcgg acgctgcgcg gctactacaa tcagagcgag gccgggtctc acaccctgca
     3961 gtggatgcat ggctgcgagc tggggcccga cgggcgcttc ctccgcgggt atgaacagtt
     4021 cgcctacgac ggcaaggatt atctcaccct gaatgaggac ctgcgctcct ggaccgcggt
     4081 ggacacggcg gctcagatct ccgagcaaaa gtcaaatgat gcctctgagg cggagcacca
     4141 gagagcctac ctggaagaca catgcgtgga gtggctccac aaatacctgg agaaggggaa
     4201 ggagacgctg cttcacctgg agcccccaaa gacacacgtg actcaccacc ccatctctga
     4261 ccatgaggcc accctgaggt gctgggccct gggcttctac cctgcggaga tcacactgac
     4321 ctggcagcag gatggggagg gccataccca ggacacggag ctcgtggaga ccaggcctgc
     4381 aggggatgga accttccaga agtgggcagc tgtggtggtg ccttctggag aggagcagag
     4441 atacacgtgc catgtgcagc atgaggggct acccgagccc gtcaccctga gatggaagcc
     4501 ggcttcccag cccaccatcc ccatcgtggg catcattgct ggcctggttc tccttggatc
     4561 tgtggtctct ggagctgtgg ttgctgctgt gatatggagg aagaagagct caggtggaaa
     4621 aggagggagc tactctaagg ctgagtggag cgacagtgtc caggggtctg agtctcacag
     4681 cttgggagga ggtggaagcg gaggaggagg aagcggagga ggaggtagct ctcgctccgt
     4741 ggccttagct gtgctcgcgc tactctctct ttctggcctg gaggctatcc agcgtactcc
     4801 aaagattcag gtttactcac gtcatccagc agagaatgga aagtcaaatt tcctgaattg
     4861 ctatgtgtct gggtttcatc catccgacat tgaagttgac ttactgaaga atggagagag
     4921 aattgaaaaa gtggagcatt cagacttgtc tttcagcaag gactggtctt tctatctctt
     4981 gtactacact gaattcaccc ccactgaaaa agatgagtat gcctgccgtg tgaaccatgt
     5041 gactttgtca cagcccaaga tagttaagtg ggatcgagac atgtaagaat tcgaagcttg
     5101 agctcgagat ctgagtccgg tagcgctagt tcgaaggatc cgcatgcatc tagggcggcc
     5161 aattccgccc ctctcccccc cccctaacgt tactggccga agccgcttgg aataaggccg
     5221 gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc
     5281 ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa
     5341 aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag
     5401 acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg
     5461 cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg
     5521 ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa
     5581 caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg
     5641 gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca
     5701 cggggacgtg gttttccttt gaaaaacacg atgataagct tgccacaacc cacaaggaga
     5761 cgaccttcca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     5821 cgggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     5881 gacccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     5941 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     6001 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     6061 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
     6121 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
     6181 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
     6241 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     6301 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
     6361 ggtgcctgat aataggcggc cgctcgagac ctagaaaaac atggagcaat cacaagtagc
     6421 aatacagcag ctaccaatgc tgattgtgcc tggctagaag cacaagagga ggaggaggtg
     6481 ggttttccag tcacacctca ggtaccttta agaccaatga cttacaaggc agctgtagat
     6541 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
     6601 caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag
     6661 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     6721 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     6781 tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc atcttattat tcagtattta
     6841 taacttgcaa agaaatgaat atcagagagt gagaggcctt gacattataa tagatttagc
     6901 aggaattgaa ctaggagtgg agcacacagg caaagttcta gagctcgctg atcagcctcg
     6961 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     7021 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     7081 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     7141 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     7201 agaaccagct gggggcgcgc ccctcgaggc cgccatggtc atagctgttt gacgtcaggt
     7261 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     7321 aatatgtatc cgctcacgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     7381 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     7441 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     7501 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     7561 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     7621 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     7681 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     7741 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     7801 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     7861 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     7921 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     7981 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     8041 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     8101 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     8161 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     8221 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     8281 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     8341 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     8401 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     8461 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     8521 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     8581 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     8641 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     8701 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     8761 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     8821 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     8881 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     8941 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     9001 tggaaaaacg ccagcaacgc ggccttttta cggttctggc cttttgca
//
