LOCUS       sequence-485599-        9158 bp    DNA     circular SYN 09-SEP-2025
DEFINITION  Empty MCP-fusion lentiviral construct for generating MCP-tethering 
            RBP libraries in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9158)
  AUTHORS   Thurm AR, Finkel Y, Andrews C, Cai XS, Benko C, Bintu L
  TITLE     High-throughput discovery of regulatory effector domains in human 
            RNA-binding proteins.
  JOURNAL   bioRxiv [Preprint]. 2024 Jul 19:2024.07.19.604317. doi: 
            10.1101/2024.07.19.604317.
  PUBMED    39071298
REFERENCE   2  (bases 1 to 9158)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 9, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Jul 19:2024.07.19.604317. doi: 
            10.1101/2024.07.19.604317."
COMMENT     Sequence Label: pAT031
FEATURES             Location/Qualifiers
     source          1..9158
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    100..225
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    722..955
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1140..1184
                     /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    1482..1599
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1722..1933
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          1934..2872
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2829..2849
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3624..3689
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3741..3794
                     /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             4521..4577
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4578..4976
                     /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(4996..5029)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5085..5673
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5138..5158)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5556..5567)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5676..5692)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5677..5693)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6202..6382
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6444..7032)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6524..6543)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7203..7994,7995..8063))
                     /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     7826..7845
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8064..8168)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8243..8262)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        8434..8813
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        8815..9013
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8968..8988
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             join(9031..9158,1..53)
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc
       61 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg
      121 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
      181 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
      241 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
      301 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
      361 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
      421 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
      481 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
      541 agtaagacca ccgcacagca agcggccggc cgctgatctt cagacctgga ggaggagata
      601 tgagggacaa ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag
      661 gagtagcacc caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa
      721 taggagcttt gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa
      781 tgacgctgac ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt
      841 tgctgagggc tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc
      901 agctccaggc aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga
      961 tttggggttg ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga
     1021 gtaataaatc tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa
     1081 ttaacaatta cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa
     1141 agaatgaaca agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca
     1201 taacaaattg gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt
     1261 taagaatagt ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat
     1321 tatcgtttca gacccacctc ccaaccccga ggggacccga caggcccgaa ggaatagaag
     1381 aagaaggtgg agagagagac agagacagat ccattcgatt agtgaacgga tcggcactgc
     1441 gtgcgccaat tctgcagaca aatggcagta ttcatccaca attttaaaag aaaagggggg
     1501 attggggggt acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact
     1561 aaagaattac aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc
     1621 agagatccag tttggttagt accgggcccg ctctagagat cccatttgac tcaggcggcc
     1681 gcgtggataa ccgtattacc gccatgcatg tgcccgtcag tgggcagagc gcacatcgcc
     1741 cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg
     1801 gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg
     1861 gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc
     1921 cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta
     1981 tggcccttgc gtgccttgaa ttacttccac ctggctgcag tacgtgattc ttgatcccga
     2041 gcttcgggtt ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc
     2101 tcgtgcttga gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca
     2161 ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc
     2221 tgctgcgacg ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac
     2281 tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg
     2341 ttcggcgagg cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc
     2401 tggccggcct gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc
     2461 aaggctggcc cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc
     2521 tgcagggagc tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac
     2581 acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg
     2641 ggcgccgtcc aggcacctcg attagttctc gcgcttttgg agtacgtcgt ctttaggttg
     2701 gggggagggg ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag
     2761 gccagcttgg cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg
     2821 gttcattctc aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg
     2881 agctagccgt taaaatggct tctaacttta ctcagttcgt tctcgtcgac aatggcggaa
     2941 ctggcgacgt gactgtcgcc ccaagcaact tcgctaacgg gatcgctgaa tggatcagct
     3001 ctaactcgcg ttcacaggct tacaaagtaa cctgtagcgt tcgtcagagc tctgcgcaga
     3061 atcgcaaata caccatcaaa gtcgaggtgc ctaaaggcgc ctggcgttcg tacttaaata
     3121 tggaactaac cattccaatt ttcgccacga attccgactg cgagcttatt gttaaggcaa
     3181 tgcaaggtct cctaaaagat ggaaacccga ttccctcagc aatcgcagca aactccggca
     3241 tctacgcgat ctacgccatg gcttctatgg cttctaactt tactcagttc gttctcgtcg
     3301 acaatggcgg aactggcgac gtgactgtcg ccccaagcaa cttcgctaac gggatcgctg
     3361 aatggatcag ctctaactcg cgttcacagg cttacaaagt aacctgtagc gttcgtcaga
     3421 gctctgcgca gaatcgcaaa tacaccatca aagtcgaggt gcctaaaggc gcctggcgtt
     3481 cgtacttaaa tatggaacta accattccaa ttttcgccac gaattccgac tgcgagctta
     3541 ttgttaaggc aatgcaaggt ctcctaaaag atggaaaccc gattccctca gcaatcgcag
     3601 caaactccgg catctacgcg atcgactaca aagaccatga cggtgattat aaagatcatg
     3661 acatcgacta caaggatgac gatgacaagg gtggatccgg atcaggctcc ggagacgggt
     3721 caagcgtctc tggatctgga gagggaaggg gaagcctcct aacttgcgga gatgtcgagg
     3781 agaatcctgg cccaatggtt agtaagggag aggagttgat aaaagagaat atgcacatga
     3841 aactgtatat ggaagggaca gtggacaacc atcacttcaa gtgcacctct gagggagaag
     3901 gaaagccata cgaaggcacc cagactatgc gaattaaggt cgtggaggga gggcctctcc
     3961 ccttcgcctt cgatattctt gcaacatctt tcctctacgg gagcaaaacc ttcatcaatc
     4021 acacacaagg catacctgac ttttttaagc agagttttcc agagggcttt acctgggagc
     4081 gtgtgactac atacgaagat gggggtgtgc tgacggctac acaggatact tcacttcagg
     4141 acggttgtct gatctacaac gtcaagatca ggggggtcaa tttcacctcg aatggaccag
     4201 taatgcaaaa aaagaccctg ggatgggaag cttttactga aactctctat cccgccgacg
     4261 gcggcctcga aggcagaaac gatatggctt tgaagctggt tggcgggtcc catctaattg
     4321 ccaatgccaa aaccacatat aggagcaaaa agcctgcaaa gaacctgaag atgccgggtg
     4381 tgtactatgt tgactataga ttagagcgga tcaaagaggc aaataacgaa acgtacgtgg
     4441 agcagcacga ggtagccgtg gcgcgctact gcgatttacc ctccaaactg ggccataaac
     4501 tgaactgtac aggaagcgga gctactaact tcagcctgct gaagcaggct ggcgacgtgg
     4561 aagaaaaccc aggacctatg gccaagcctt tgtctcaaga agaatccacc ctcattgaaa
     4621 gagcaacggc tacaatcaac agcatcccca tctctgaaga ctacagcgtc gccagcgcag
     4681 ctctctctag cgacggccgc atcttcactg gtgtcaatgt atatcatttt actgggggac
     4741 cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc ggcagctggc aacctgactt
     4801 gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag cccctgcgga cggtgccgac
     4861 aggtgcttct cgatctgcat cctgggatca aagccatagt gaaggacagt gatggacagc
     4921 cgacggcagt tgggattcgt gaattgctgc cctctggtta tgtgtgggag ggctaagaat
     4981 tcgtcgaggg acctaataac ttcgtatagc atacattata cgaagttata catgtttaag
     5041 ggttccggtt ccactaggta caattcgata tcaagcttat cgataatcaa cctctggatt
     5101 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     5161 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     5221 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     5281 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     5341 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     5401 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     5461 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
     5521 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     5581 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     5641 cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgacct cgatcgagac
     5701 ctagaaaaac atggagcaat cacaagtagc aatacagcag ctaccaatgc tgattgtgcc
     5761 tggctagaag cacaagagga ggaggaggtg ggttttccag tcacacctca ggtaccttta
     5821 agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga
     5881 ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg gatctaccac
     5941 acacaaggct acttccctga ttggcagaac tacacaccag ggccagggat cagatatcca
     6001 ctgacctttg gatggtgcta caagctagta ccagttgagc aagagaaggt agaagaagcc
     6061 aatgaaggag agaacacccg cttgttacac cctgtgagcc tgcatgggat ggatgacccg
     6121 gagagagaag tattagagtg gaggtttgac agccgcctag catttcatca catggcccga
     6181 gagctgcatc cggactgtac tgggtctctc tggttagacc agatctgagc ctgggagctc
     6241 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa
     6301 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
     6361 tcagtgtgga aaatctctag cagcatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     6421 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     6481 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     6541 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     6601 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6661 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6721 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     6781 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6841 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6901 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6961 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     7021 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     7081 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     7141 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     7201 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     7261 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     7321 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     7381 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     7441 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     7501 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     7561 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     7621 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     7681 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     7741 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     7801 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     7861 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7921 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7981 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     8041 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     8101 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     8161 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg
     8221 atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
     8281 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
     8341 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc
     8401 tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact agttattaat
     8461 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
     8521 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
     8581 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
     8641 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
     8701 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
     8761 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
     8821 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
     8881 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
     8941 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
     9001 tctatataag cagcgcgttt tgcctgtact gggtctctct ggttagacca gatctgagcc
     9061 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     9121 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactc
//
