LOCUS       sequence-324343-        8921 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral backbone for GoldenGate cloning effectors as a fusion 
            with rTetR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8921)
  AUTHORS   Tycko J, DelRosso N, Hess GT, Aradhana, Banerjee A, Mukund A, Van 
            MV, Ego BK, Yao D, Spees K, Suzuki P, Marinov GK, Kundaje A, Bassik 
            MC, Bintu L
  TITLE     High-Throughput Discovery and Characterization of Human 
            Transcriptional Effectors.
  JOURNAL   Cell. 2020 Dec 9. pii: S0092-8674(20)31541-5. doi: 
            10.1016/j.cell.2020.11.024.
  PUBMED    33326746
REFERENCE   2  (bases 1 to 8921)
  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. 2020 
            Dec 9. pii: S0092-8674(20)31541-5. doi: 10.1016/j.cell.2020.11.024."
COMMENT     Sequence Label: pJT050 lenti
            pEF-rTetR-LibCloneSite-T2A-mCherr...
FEATURES             Location/Qualifiers
     source          1..8921
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(552..1343,1344..1412))
                     /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     1175..1194
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1413..1517)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1592..1611)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1783..2162
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2164..2362
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2317..2337
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             2380..2560
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2607..2732
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3229..3462
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3647..3691
                     /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    3989..4106
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4229..4440
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          4441..5379
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5336..5356
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             6068..6121
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             6122..6829
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(6267..6285)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6535..6554
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             6848..7246
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    complement(7266..7299)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    7355..7943
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7408..7428)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7826..7837)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7946..7962)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7947..7963)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             8472..8652
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(join(8714..8921,1..381))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8794..8813)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
       61 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
      121 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
      181 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
      241 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
      301 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
      361 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
      421 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
      481 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
      541 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
      601 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
      661 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
      721 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
      781 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
      841 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
      901 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
      961 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     1021 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     1081 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     1141 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     1201 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     1261 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     1321 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     1381 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     1441 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     1501 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
     1561 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     1621 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     1681 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
     1741 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
     1801 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     1861 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     1921 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     1981 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     2041 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     2101 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     2161 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
     2221 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
     2281 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
     2341 ggtgggaggt ctatataagc agcgcgtttt gcctgtactg ggtctctctg gttagaccag
     2401 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
     2461 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
     2521 tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact
     2581 tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc
     2641 gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag
     2701 gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc
     2761 gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag
     2821 tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag
     2881 aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac
     2941 ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa
     3001 aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg
     3061 cacagcaagc ggccggccgc tgatcttcag acctggagga ggagatatga gggacaattg
     3121 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac
     3181 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt
     3241 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt
     3301 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat
     3361 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag
     3421 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc
     3481 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct
     3541 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac
     3601 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga
     3661 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct
     3721 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt
     3781 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac
     3841 ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga
     3901 gagagacaga gacagatcca ttcgattagt gaacggatcg gcactgcgtg cgccaattct
     3961 gcagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca
     4021 gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa
     4081 aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga gatccagttt
     4141 ggttagtacc gggcccgctc tagagatccc atttgactca ggcggccgcg tggataaccg
     4201 tattaccgcc atgcatgtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag
     4261 aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac
     4321 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     4381 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     4441 gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg
     4501 ccttgaatta cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga
     4561 agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt
     4621 gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt
     4681 ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt
     4741 tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt
     4801 tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg
     4861 ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct
     4921 ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg
     4981 tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca
     5041 aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg
     5101 gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg
     5161 cacctcgatt agttctcgcg cttttggagt acgtcgtctt taggttgggg ggaggggttt
     5221 tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac
     5281 ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag
     5341 cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgagc tagccaccat
     5401 gtctagactg gacaagagca aagtcataaa cggagctctg gaattactca atggtgtcgg
     5461 tatcgaaggc ctgacgacaa ggaaactcgc tcaaaagctg ggagttgagc agcctaccct
     5521 gtactggcac gtgaagaaca agcgggccct gctcgatgcc ctgccaatcg agatgctgga
     5581 caggcatcat acccacttct gccccctgga aggcgagtca tggcaagact ttctgcggaa
     5641 caacgccaag tcataccgct gtgctctcct ctcacatcgc gacggggcta aagtgcatct
     5701 cggcacccgc ccaacagaga aacagtacga aaccctggaa aatcagctcg cgttcctgtg
     5761 tcagcaaggc ttctccctgg agaacgcact gtacgctctg tccgccgtgg gccactttac
     5821 actgggctgc gtattggagg aacaggagca tcaagtagca aaagaggaaa gagagacacc
     5881 taccaccgat tctatgcccc cacttctgag acaagcaatt gagctgttcg accggcaggg
     5941 agccgaacct gccttccttt tcggcctgga actaatcata tgtggcctgg agaaacagct
     6001 aaagtgcgaa agcggcggtg gatccggatc aggctccgga gacgggtcaa gcgtctctgg
     6061 atctggagag ggaaggggaa gcctcctaac ttgcggagat gtcgaggaga atcctggccc
     6121 aatggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa
     6181 ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
     6241 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc
     6301 cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca
     6361 ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg
     6421 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
     6481 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt
     6541 aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg
     6601 cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc
     6661 tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt
     6721 caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga
     6781 acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagg gtagcggcag
     6841 cggtagcatg gccaagcctt tgtctcaaga agaatccacc ctcattgaaa gagcaacggc
     6901 tacaatcaac agcatcccca tctctgaaga ctacagcgtc gccagcgcag ctctctctag
     6961 cgacggccgc atcttcactg gtgtcaatgt atatcatttt actgggggac cttgtgcaga
     7021 actcgtggtg ctgggcactg ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc
     7081 gatcggaaat gagaacaggg gcatcttgag cccctgcgga cggtgccgac aggtgcttct
     7141 cgatctgcat cctgggatca aagccatagt gaaggacagt gatggacagc cgacggcagt
     7201 tgggattcgt gaattgctgc cctctggtta tgtgtgggag ggctaagaat tcgtcgaggg
     7261 acctaataac ttcgtatagc atacattata cgaagttata catgtttaag ggttccggtt
     7321 ccactaggta caattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg
     7381 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     7441 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     7501 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     7561 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     7621 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     7681 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     7741 gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg
     7801 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     7861 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     7921 ctccctttgg gccgcctccc cgcatcgata ccgtcgacct cgatcgagac ctagaaaaac
     7981 atggagcaat cacaagtagc aatacagcag ctaccaatgc tgattgtgcc tggctagaag
     8041 cacaagagga ggaggaggtg ggttttccag tcacacctca ggtaccttta agaccaatga
     8101 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
     8161 taattcactc ccaacgaaga caagatatcc ttgatctgtg gatctaccac acacaaggct
     8221 acttccctga ttggcagaac tacacaccag ggccagggat cagatatcca ctgacctttg
     8281 gatggtgcta caagctagta ccagttgagc aagagaaggt agaagaagcc aatgaaggag
     8341 agaacacccg cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag
     8401 tattagagtg gaggtttgac agccgcctag catttcatca catggcccga gagctgcatc
     8461 cggactgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
     8521 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     8581 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     8641 aaatctctag cagcatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     8701 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     8761 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     8821 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     8881 tcccttcggg aagcgtggcg ctttctcata gctcacgctg t
//
