LOCUS       sequence-321900-        8387 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  lentiviral overexpression vector for human Rap2A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8387)
  AUTHORS   Rossiter NJ, Huggler KS, Adelmann CH, Keys HR, Soens RW, Sabatini 
            DM, Cantor JR
  TITLE     CRISPR screens in physiologic medium reveal conditionally essential 
            genes in human cells.
  JOURNAL   Cell Metab. 2021 Feb 23. pii: S1550-4131(21)00061-9. doi: 
            10.1016/j.cmet.2021.02.005.
  PUBMED    33651980
REFERENCE   2  (bases 1 to 8387)
  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: "Cell Metab.
            2021 Feb 23. pii: S1550-4131(21)00061-9. doi: 
            10.1016/j.cmet.2021.02.005."
COMMENT     Sequence Label: pLJC6-Rap2A-3xFLAG
FEATURES             Location/Qualifiers
     source          1..8387
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     123..142
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     376..393
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    510..531
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(546..551,552..569,570..576)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    584..600
                     /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     589..611
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     608..624
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     608..624
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     643..663
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        645..663
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        691..917
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             918..1098
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1145..1270
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1763..1996
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2181..2225
                     /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        2514..3725
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3562..3581
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4331..4396
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    4444..4561
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4610..5120
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     5039..5057
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             5129..5524
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MVPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             5596..5829
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5901..6022
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5938..5957)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5992..6011
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6062..6197
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6217..6236)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6218..6236)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6246..6263)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6246..6262)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6255..6277)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6404..6859
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6491..6510)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6701..6722
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6885..6989
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6990..7058,7059..7850)
                     /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(7208..7227)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(8021..8387,1..222)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
       61 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
      121 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
      181 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
      241 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
      301 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
      361 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
      421 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
      481 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
      541 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
      601 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
      661 agggaacaaa agctggagct gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt
      721 gcaacatggt aacgatgagt tagcaacatg ccttacaagg agagaaaaag caccgtgcat
      781 gccgattggt ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa cagacgggtc
      841 tgacatggat tggacgaacc actgaattgc cgcattgcag agatattgta tttaagtgcc
      901 tagctcgata cataaacggg tctctctggt tagaccagat ctgagcctgg gagctctctg
      961 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
     1021 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     1081 tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga
     1141 ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg
     1201 gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg
     1261 cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag
     1321 gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga
     1381 acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg
     1441 acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt
     1501 agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga
     1561 caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcag cagctgatct
     1621 tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat aaatataaag
     1681 tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga
     1741 gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa
     1801 gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta
     1861 tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac
     1921 tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa
     1981 aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg
     2041 tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct
     2101 ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag
     2161 aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa
     2221 gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga
     2281 tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag
     2341 ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg aggggacccg
     2401 acaggcccga aggaatagaa gaagaaggtg gagagagaga cagagacaga tccattcgat
     2461 tagtgaacgg atctcgacgg tatcgatcac gagactagcc tcgagcggga tctggcctcc
     2521 gcgccgggtt ttggcgcctc ccgcgggcgc ccccctcctc acggcgagcg ctgccacgtc
     2581 agacgaaggg cgcagcgagc gtcctgatcc ttccgcccgg acgctcagga cagcggcccg
     2641 ctgctcataa gactcggcct tagaacccca gtatcagcag aaggacattt taggacggga
     2701 cttgggtgac tctagggcac tggttttctt tccagagagc ggaacaggcg aggaaaagta
     2761 gtcccttctc ggcgattctg cggagggatc tccgtggggc ggtgaacgcc gatgattata
     2821 taaggacgcg ccgggtgtgg cacagctagt tccgtcgcag ccgggatttg ggtcgcggtt
     2881 cttgtttgtg gatcgctgtg atcgtcactt ggtgagtagc gggctgctgg gctggccggg
     2941 gctttcgtgg ccgccgggcc gctcggtggg acggaagcgt gtggagagac cgccaagggc
     3001 tgtagtctgg gtccgcgagc aaggttgccc tgaactgggg gttgggggga gcgcagcaaa
     3061 atggcggctg ttcccgagtc ttgaatggaa gacgcttgtg aggcgggctg tgaggtcgtt
     3121 gaaacaaggt ggggggcatg gtgggcggca agaacccaag gtcttgaggc cttcgctaat
     3181 gcgggaaagc tcttattcgg gtgagatggg ctggggcacc atctggggac cctgacgtga
     3241 agtttgtcac tgactggaga actcggtttg tcgtctgttg cgggggcggc agttatggcg
     3301 gtgccgttgg gcagtgcacc cgtacctttg ggagcgcgcg ccctcgtcgt gtcgtgacgt
     3361 cacccgttct gttggcttat aatgcagggt ggggccacct gccggtaggt gtgcggtagg
     3421 cttttctccg tcgcaggacg cagggttcgg gcctagggta ggctctcctg aatcgacagg
     3481 cgccggacct ctggtgaggg gagggataag tgaggcgtca gtttctttgg tcggttttat
     3541 gtacctatct tcttaagtag ctgaagctcc ggttttgaac tatgcgctcg gggttggcga
     3601 gtgtgttttg tgaagttttt taggcacctt ttgaaatgta atcatttggg tcaatatgta
     3661 attttcagtg ttagactagt aaattgtccg ctaaattctg gccgtttttg gcttttttgt
     3721 tagacgaagc gctagcgcta ccggtttaat taaagggacg atgcgcgagt acaaagtggt
     3781 ggtgctgggc tcgggcgggg taggcaaatc cgccctgacc gtgcagttcg tgaccggcac
     3841 cttcatcgag aaatacgacc ccaccatcga ggacttctac cgcaaggaga tcgaggtgga
     3901 ttcgtcgccg tcggtgctgg agatcctgga cacggcgggc accgagcagt tcgcgtccat
     3961 gcgggacctg tacatcaaga acggccaggg cttcatcctc gtctacagcc tcgtcaacca
     4021 gcagagcttc caggacatca agcccatgcg ggaccagatc atccgcgtga agcggtatga
     4081 gaaagtgcca gtcatcttgg ttgggaacaa agtggacctg gaaagtgaga gagaagtatc
     4141 gtccagcgaa ggcagagccc ttgctgaaga gtggggctgc ccctttatgg aaacttccgc
     4201 taagagtaaa acaatggtgg acgaactctt tgcagaaatt gtgaggcaga tgaactatgc
     4261 tgctcagcct gacaaagatg acccatgctg ttctgcatgt aacatacaag gaggcagcgc
     4321 ggccgcaggc gactacaaag accatgacgg tgattataaa gatcatgaca ttgattacaa
     4381 ggatgacgat gacaagtaat aagaattctc gacctcgaga caaatggcag tattcatcca
     4441 caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     4501 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
     4561 tcgggtttat tacagggaca gcagagatcc actttggccg cggctcgagg gggttggggt
     4621 tgcgcctttt ccaaggcagc cctgggtttg cgcagggacg cggctgctct gggcgtggtt
     4681 ccgggaaacg cagcggcgcc gaccctgggt ctcgcacatt cttcacgtcc gttcgcagcg
     4741 tcacccggat cttcgccgct acccttgtgg gccccccggc gacgcttcct gctccgcccc
     4801 taagtcggga aggttccttg cggttcgcgg cgtgccggac gtgacaaacg gaagccgcac
     4861 gtctcactag taccctcgca gacggacagc gccagggagc aatggcagcg cgccgaccgc
     4921 gatgggctgt ggccaatagc ggctgctcag cagggcgcgc cgagagcagc ggccgggaag
     4981 gggcggtgcg ggaggcgggg tgtggggcgg tagtgtgggc cctgttcctg cccgcgcggt
     5041 gttccgcatt ctgcaagcct ccggagcgca cgtcggcagt cggctccctc gttgaccgaa
     5101 tcaccgacct ctctccccag ggggatccat ggttcctttg tctcaagaag aatccaccct
     5161 cattgaaaga gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc
     5221 cagcgcagct ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac
     5281 tgggggacct tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa
     5341 cctgacttgt atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg
     5401 gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa gccatagtga aggacagtga
     5461 tggacagccg acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg
     5521 ctaaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     5581 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg
     5641 cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     5701 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     5761 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     5821 tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat
     5881 gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca
     5941 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     6001 ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc
     6061 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     6121 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     6181 aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg
     6241 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     6301 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     6361 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc
     6421 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     6481 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     6541 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     6601 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     6661 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     6721 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     6781 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     6841 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     6901 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     6961 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     7021 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     7081 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     7141 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     7201 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     7261 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     7321 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     7381 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     7441 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     7501 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     7561 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     7621 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     7681 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     7741 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     7801 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     7861 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     7921 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     7981 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     8041 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     8101 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     8161 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     8221 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     8281 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     8341 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacacc
//
