LOCUS       sequence-308776-        8354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression of a customizable open reading frame.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8354)
  AUTHORS   Hanna RE, Hegde M, Fagre CR, DeWeirdt PC, Sangree AK, Szegletes Z, 
            Griffith A, Feeley MN, Sanson KR, Baidi Y, Koblan LW, Liu DR, Neal 
            JT, Doench JG
  TITLE     Massively parallel assessment of human variants with base editor 
            screens.
  JOURNAL   Cell. 2021 Feb 18;184(4):1064-1080.e20. doi: 
            10.1016/j.cell.2021.01.012.
  PUBMED    33606977
REFERENCE   2  (bases 1 to 8354)
  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"; doi: 
            "10.1016/j.cell.2021.01"; journalName: "Cell"; date: "2021-02-18- 
            18"; volume: "184"; issue: "4"; pages: "1064-1080.e20"
COMMENT     Sequence Label: pMT_025
FEATURES             Location/Qualifiers
     source          1..8354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    41..158
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        296..1474
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          527..1465
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1422..1442
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     misc_feature    1587..2175
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1640..1660)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2058..2069)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2247..2480
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2558..2679
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2595..2614)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2649..2668
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2719..2854
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2874..2893)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2875..2893)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2903..2920)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2903..2919)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2912..2934)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3061..3516
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3148..3167)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3358..3379
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3542..3646
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3647..3715,3716..4507)
                     /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(3865..3884)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4678..5266
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5167..5186
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5420..5437
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5554..5575
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5590..5595,5596..5613,5614..5620)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5628..5644
                     /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     5633..5655
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5652..5668
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5652..5668
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5687..5707
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5689..5707
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5735..5961
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5962..6142
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6189..6314
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6807..7040
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7225..7269
                     /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        7500..7710
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        7522..7718
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      7569..7704
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             7750..8352
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGAS"
     primer_bind     complement(7750..7769)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     8246..8266
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
ORIGIN
        1 aattctagat cttgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
       61 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
      121 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
      181 gagatccact ttggcgccgg ctcgaggcct gcaggtgcaa agatggataa agttttaaac
      241 agagaggaat ctttgcagct aatggacctt ctaggtcttg aaaggagtgg gaattggctc
      301 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
      361 ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
      421 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
      481 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg
      541 tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt gaattacttc
      601 cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg ggtgggagag
      661 ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg cctggcctgg
      721 gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg ctgctttcga
      781 taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt tctggcaaga
      841 tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg gggccgcggg
      901 cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg
      961 gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg tgcctggcct
     1021 cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg caccagttgc
     1081 gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg
     1141 gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc
     1201 agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc tcgattagtt
     1261 ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg cgatggagtt
     1321 tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga tgtaattctc
     1381 cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc agacagtggt
     1441 tcaaagtttt tttcttccat ttcaggtgtc gtgaggctag ccatatgatc gattgatcag
     1501 cattctacga cgtggctgtc agctcctaag acaacttgcg caagaagggg atatcggatc
     1561 ccgggactag tacgcgttaa gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat
     1621 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     1681 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     1741 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     1801 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     1861 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     1921 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     1981 aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
     2041 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     2101 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     2161 gggccgcctc cccgcgtcga ctttaagacc aatgacttac aaggcagctg tagatcttag
     2221 ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga
     2281 tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
     2341 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
     2401 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
     2461 agtgtggaaa atctctagca gtacgtatag tagttcatgt catcttatta ttcagtattt
     2521 ataacttgca aagaaatgaa tatcagagag tgagaggaac ttgtttattg cagcttataa
     2581 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     2641 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggc tctagctatc
     2701 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     2761 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     2821 ttccagaagt agtgaggagg cttttttgga ggcctaggga cgtacccaat tcgccctata
     2881 gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     2941 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     3001 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg
     3061 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     3121 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3181 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     3241 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     3301 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     3361 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     3421 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     3481 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     3541 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     3601 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     3661 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     3721 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     3781 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     3841 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     3901 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     3961 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     4021 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     4081 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     4141 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     4201 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     4261 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     4321 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     4381 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     4441 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     4501 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     4561 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     4621 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     4681 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     4741 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     4801 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     4861 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4921 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     4981 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5041 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5101 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5161 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5221 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     5281 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     5341 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     5401 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     5461 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     5521 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
     5581 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
     5641 acaatttcac acaggaaaca gctatgacca tgattacgcc aagcgcgcaa ttaaccctca
     5701 ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt cttatgcaat actcttgtag
     5761 tcttgcaaca tggtaacgat gagttagcaa catgccttac aaggagagaa aaagcaccgt
     5821 gcatgccgat tggtggaagt aaggtggtac gatcgtgcct tattaggaag gcaacagacg
     5881 ggtctgacat ggattggacg aaccactgaa ttgccgcatt gcagagatat tgtatttaag
     5941 tgcctagctc gatacataaa cgggtctctc tggttagacc agatctgagc ctgggagctc
     6001 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa
     6061 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
     6121 tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac
     6181 cagaggagct ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg
     6241 ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg
     6301 ggtgcgagag cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt
     6361 taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc
     6421 tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac
     6481 tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata
     6541 cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt
     6601 tagacaagat agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg
     6661 atcttcagac ctggaggagg agatatgagg gacaattgga gaagtgaatt atataaatat
     6721 aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg
     6781 cagagagaaa aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca
     6841 ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct
     6901 ggtatagtgc agcagcagaa caatttgctg agggctattg aggcgcaaca gcatctgttg
     6961 caactcacag tctggggcat caagcagctc caggcaagaa tcctggctgt ggaaagatac
     7021 ctaaaggatc aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact
     7081 gctgtgcctt ggaatgctag ttggagtaat aaatctctgg aacagatttg gaatcacacg
     7141 acctggatgg agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt
     7201 gaagaatcgc aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg
     7261 gcaagtttgt ggaattggtt taacataaca aattggctgt ggtatataaa attattcata
     7321 atgatagtag gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat
     7381 agagttaggc agggatattc accattatcg tttcagaccc acctcccaac cccgagggga
     7441 cccatgcatt gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     7501 caggcagaag tatgcaaagc atgcgtctca attagtcagc aaccatagtc ccgcccctaa
     7561 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     7621 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     7681 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctttctaga ggtaccaccg
     7741 gagcttacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     7801 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     7861 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     7921 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgccgtggc ggtctggacc
     7981 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     8041 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
     8101 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
     8161 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
     8221 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     8281 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
     8341 ggtgccagtt aacg
//
