LOCUS       sequence-455143-        6059 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1 attB_GA mCherry-T2A-puro, Dox-inducible EYFP reporter for 
            integration into Bxb1 attP_GA landing pad.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6059)
  AUTHORS   Nugent PJ, Park H, Wladyka CL, Yelland JN, Sinha S, Chen KY, Bynum 
            C, Quarterman G, Lee SC, Hsieh AC, Subramaniam AR
  TITLE     Decoding post-transcriptional regulatory networks by RNA-linked 
            CRISPR screening in human cells.
  JOURNAL   Nat Methods. 2025 Jun;22(6):1237-1246. doi: 
            10.1038/s41592-025-02702-6. Epub 2025 May 29.
  PUBMED    40442371
REFERENCE   2  (bases 1 to 6059)
  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: 
            "10.1038/s41592-025-02702-6"; journalName: "Nat Methods"; date: 
            "2025-06"; volume: "22"; issue: "6"; pages: "1237-1246"
COMMENT     Sequence Label: pHPHS853
FEATURES             Location/Qualifiers
     source          1..6059
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1259..1611
                     /label=TRE3GV promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, optimized for 
                     retroviral and lentiviral vectors"
     protein_bind    1267..1285
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1303..1321
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1339..1357
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1375..1393
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1411..1429
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1447..1465
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1483..1501
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             join(1624..1626,1627..1629,1630..2340)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1669..1690)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1930..1949)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2277..2298
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2341..2367
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    2621..2658
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     regulatory      2667..2676
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2673..3380
                     /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(2818..2836)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3086..3105
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3381..3434
                     /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             3438..4037
                     /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(3438..3457)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3934..3954
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4094..4113)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4148..4167
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(4304..5095,5096..5164))
                     /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     4927..4946
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5165..5269)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5372..5960
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5861..5880
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcggccgcga gctcacgggg acagcccccc cccaaagccc ccagggatgt aattacgtcc
       61 ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc ggcgctcccc
      121 ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg tggcacggga
      181 tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg gggggatacg
      241 gggaaaaagc tttaggctga aagagagatt tagaatgaca gaatcataga acggcctggg
      301 ttgcaaagga gcacagtgct catccagatc caaccccctg ctatgtgcag ggtcatcaac
      361 cagcagccca ggctgcccag agccacatcc agcctggcct tgaatgcctg cagggatggg
      421 gcatccacag cctccttggg caacctgttc agtgcgtcac caccctctgg gggaaaaact
      481 gcctcctcat atccaaccca aacctcccct gtctcagtgt aaagccattc ccccttgtcc
      541 tatcaagggg gagtttgctg tgacattgtt ggtctggggt gacacatgtt tgccaattca
      601 gtgcatcacg gagaggcaga tcttggggat aaggaagtgc aggacagcat ggacgtggga
      661 catgcaggtg ttgagggctc tgggacactc tccaagtcac agcgttcaga acagccttaa
      721 ggataagaag ataggataga aggacaaaga gcaagttaaa acccagcatg gagaggagca
      781 caaaaaggcc acagacactg ctggtccctg tgtctgagcc tgcatgtttg atggtgtctg
      841 gatgcaagca gaaggggtgg aagagcttgc ctggagagat acagctgggt cagtaggact
      901 gggacaggca gctggagaat tgccatgtag atgttcatac aatcgtcaaa tcatgaaggc
      961 tggaaaagcc ctccaagatc cccaagacca accccaaccc acccaccgtg cccactggcc
     1021 atgtccctca gtgccacatc cccacagttc ttcatcacct ccagggacgg tgaccccccc
     1081 acctccgtgg gcagctgtgc cactgcagca ccgctctttg gagaaggtaa atcttgctaa
     1141 atccagcccg accctcccct ggcacaacgt aaggccatta tctctcatcc aactccagga
     1201 cggagtcagt gagaatattg gcgcgccgat atccgatgag gccctttcgt cttcactcga
     1261 gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc agtgatagag
     1321 aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt ttactcccta
     1381 tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa cgtatctaca
     1441 gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc agtgatagag
     1501 aacgtatgtc gaggtaggcg tgtacggtgg gcgcctataa aagcagagct cgtttagtga
     1561 accgaattca gatcgcctgg agcaattcca caacactttt gtcttatact taagcttggt
     1621 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     1681 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     1741 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     1801 accctcgtga ccaccttcgg ctacggcctg cagtgcttcg cccgctaccc cgaccacatg
     1861 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     1921 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     1981 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     2041 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     2101 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     2161 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     2221 cactacctga gctaccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     2281 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     2341 tacccatacg atgttccgga ttacgcttag gccaatggaa agatcgccgt gtaagcggcc
     2401 gcctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     2461 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2521 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2581 caatagcagg catgctgggg atgcggtggg ctctatggcc ggcttgtcga cgacggcgga
     2641 ctccgtcgtc aggatcatcc gcgctagcca ccatggtgag caagggcgag gaggataaca
     2701 tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc
     2761 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
     2821 agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt
     2881 tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc
     2941 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg
     3001 tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc
     3061 gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     3121 cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc
     3181 tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga
     3241 agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca
     3301 acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg
     3361 gcatggacga gctgtacaag gagggcagag gaagtcttct aacatgcggt gacgtggagg
     3421 agaatcccgg ccctaggatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
     3481 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
     3541 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
     3601 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
     3661 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     3721 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     3781 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtcagc cccgaccacc
     3841 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     3901 gggtgcccgc cttcctggag acatccgcgc cccgcaacct ccccttctac gagcggctcg
     3961 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     4021 gcaagcccgg tgcctgagcc aatccatcag agagatcctc gcggccgcgg atccagacat
     4081 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     4141 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaact
     4201 ctagagagtg attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     4261 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     4321 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     4381 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     4441 cgcgggaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     4501 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     4561 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     4621 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     4681 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     4741 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     4801 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     4861 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     4921 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     4981 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     5041 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     5101 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     5161 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     5221 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     5281 gaaaagtgcc acctgtcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     5341 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     5401 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     5461 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     5521 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     5581 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     5641 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5701 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     5761 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     5821 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     5881 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5941 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     6001 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgt
//
