LOCUS       sequence-273175-        9028 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpression of HLA-G fused to B2M.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9028)
  TITLE     Lian Lab CRISPR plasmids
REFERENCE   2  (bases 1 to 9028)
  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: pLR03_pXL001-_B2MHLAG
FEATURES             Location/Qualifiers
     source          1..9028
                     /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"
     CDS             complement(3987..4013)
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHYHHHH"
     misc_feature    join(5144..5718,5719..5721,5722..5730)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5311..5328)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5538..5557
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5750..6349
                     /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(5750..5769)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6246..6266
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             6552..6785
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6936..6953)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6942..7166
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     regulatory      7209..7218
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        7262..7366
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7367..7435,7436..8227)
                     /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(7585..7604)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8398..8986
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8887..8906
                     /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 gccaccatgt ctcgctccgt ggccttagct gtgctcgcgc tactctctct
     3661 ttctggcctg gaggctatcc agcgtactcc aaagattcag gtttactcac gtcatccagc
     3721 agagaatgga aagtcaaatt tcctgaattg ctatgtgtct gggtttcatc catccgacat
     3781 tgaagttgac ttactgaaga atggagagag aattgaaaaa gtggagcatt cagacttgtc
     3841 tttcagcaag gactggtctt tctatctctt gtactacact gagttcaccc ccactgaaaa
     3901 agatgagtat gcctgccgtg tgaaccatgt gactttgtca cagcccaaga tagttaagtg
     3961 ggatcgagac atgggtggtg gtggtagtgg tggtggtggt agtggtggtg gtggtagtaa
     4021 gacgccaagg atggtggtca tggcgccccg aaccctcttc ctgctgctct cgggggccct
     4081 gaccctgacc gagacctggg cgggctccca ctccatgagg tatttcagcg ccgccgtgtc
     4141 ccggcccggc cgcggggagc cccgcttcat cgccatgggc tacgtggacg acacgcagtt
     4201 cgtgcggttc gacagcgact cggcgtgtcc gaggatggag ccgcgggcgc cgtgggtgga
     4261 gcaggagggg ccggagtatt gggaagagga gacacggaac accaaggccc acgcacagac
     4321 tgacagaatg aacctgcaga ccctgcgcgg ctactacaac cagagcgagg ccagttctca
     4381 caccctccag tggatgattg gctgcgacct ggggtccgac ggacgcctcc tccgcgggta
     4441 tgaacagtat gcctacgatg gcaaggatta cctcgccctg aacgaggacc tgcgctcctg
     4501 gaccgcagcg gacactgcgg ctcagatctc caagcgcaag tgtgaggcgg ccaatgtggc
     4561 tgaacaaagg agagcctacc tggagggcac gtgcgtggag tggctccaca gatacctgga
     4621 gaacgggaag gagatgctgc agcgcgcgga cccccccaag acacacgtga cccaccaccc
     4681 tgtctttgac tatgaggcca ccctgaggtg ctgggccctg ggcttctacc ctgcggagat
     4741 catactgacc tggcagcggg atggggagga ccagacccag gacgtggagc tcgtggagac
     4801 caggcctgca ggggatggaa ccttccagaa gtgggcagct gtggtggtgc cttctggaga
     4861 ggagcagaga tacacgtgcc atgtgcagca tgaggggctg ccggagcccc tcatgctgag
     4921 atggaagcag tcttccctgc ccaccatccc catcatgggt atcgttgctg gcctggttgt
     4981 ccttgcagct gtagtcactg gagctgcggt cgctgctgtg ctgtggagaa agaagagctc
     5041 agattgagaa ttcgaagctt gagctcgaga tctgagtccg gtagcgctag ttcgaaggat
     5101 ccgcatgcat ctagggcggc caattccgcc cctctccccc ccccccctct ccctcccccc
     5161 cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt
     5221 tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct
     5281 tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga
     5341 atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga
     5401 ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac
     5461 gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag
     5521 ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc
     5581 agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg
     5641 tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt
     5701 gaaaaacacg atgataagct tgccacaacc cacaaggaga cgaccttcca tgaccgagta
     5761 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc cgggccgtac gcaccctcgc
     5821 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gacccggacc gccacatcga
     5881 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt
     5941 gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc
     6001 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc
     6061 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt
     6121 cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt
     6181 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc
     6241 gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt
     6301 gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgat aataggcggc
     6361 cgctcgagac ctagaaaaac atggagcaat cacaagtagc aatacagcag ctaccaatgc
     6421 tgattgtgcc tggctagaag cacaagagga ggaggaggtg ggttttccag tcacacctca
     6481 ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga
     6541 aaagggggga ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg
     6601 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
     6661 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
     6721 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
     6781 tagcagtagt agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat
     6841 atcagagagt gagaggcctt gacattataa tagatttagc aggaattgaa ctaggagtgg
     6901 agcacacagg caaagttcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     6961 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     7021 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     7081 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     7141 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct gggggcgcgc
     7201 ccctcgaggc cgccatggtc atagctgttt gacgtcaggt ggcacttttc ggggaaatgt
     7261 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcacgag
     7321 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     7381 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     7441 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     7501 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     7561 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     7621 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     7681 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     7741 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     7801 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     7861 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     7921 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     7981 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     8041 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     8101 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     8161 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     8221 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     8281 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     8341 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     8401 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     8461 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     8521 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     8581 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     8641 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     8701 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     8761 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     8821 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     8881 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     8941 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     9001 ggccttttta cggttctggc cttttgca
//
