LOCUS       sequence-455212-        6641 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1 attB_GA mCherry-T2A-puro, Dox-inducible beta-globin NMD 
            reporter with premature termination codon for integration into Bxb1 
            attP_GA landing pad.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6641)
  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 6641)
  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: pHPHS926
FEATURES             Location/Qualifiers
     source          1..6641
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1259..1608
                     /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"
     primer_bind     1547..1571
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1615..1624
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1624..1689
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2923..2949
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    3203..3240
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     regulatory      3249..3258
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3255..3962
                     /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(3400..3418)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3668..3687
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3963..4016
                     /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             4020..4619
                     /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(4020..4039)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4516..4536
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4676..4695)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4730..4749
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(4886..5677,5678..5746))
                     /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     5509..5528
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5747..5851)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5954..6542
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6443..6462
                     /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 accgtcagat cgcctggagc aattccacaa cacttttgtc ttatacttaa gcttgccacc
     1621 atggactaca aggaccacga cggcgattat aaggatcacg acatcgacta caaagacgac
     1681 gatgacaagg tcgacgtgca tctgactcct gaggagaagt ctgccgttac tgccctgtgg
     1741 ggcaaggtga acgtggatga agttggtggt gaggccctgg gcaggttggt atcaaggtta
     1801 caagacaggt ttaaggagac caatagaaac tgggcatgtg gagacagaga agactcttgg
     1861 gtttctgata ggcactgact ctctctgcca attggtctat tttcccaccc ttaggctgct
     1921 ggtggtctac ccttggacct agaggttctt tgagtccttt gggatctgtc cactcctgat
     1981 gctgttatgg gcaaccctaa ggtgaaggct catggcaaga aagtgctcgg tgcctttagt
     2041 gatggcctgg ctcacctgga caacctcaag ggcacctttg ccacactgag tgagctgcac
     2101 tgtgacaagc tgcacgtgga tcctgagaac ttcagggtga gtctgatggg cacctcctgg
     2161 gtttccttcc cctggctatt ctgctcaacc ttcctatcag aaaaaaaggg gaagcgattc
     2221 tagggagcag tctccatgac tgtgtgtgga gtgttgacaa gagttcggat attttattct
     2281 ctactcagaa ttgctgctcc ccctcactct gttctgtgtt gtcatttcct ctttctttgg
     2341 taagctttta atttccagtt gcattttact aaattaatta agctggttat ttacttccca
     2401 tcctgatatc agcttcccct cctcctttcc tcccagtcct tctctctctc ctctctcttt
     2461 ctctaatcct ttcctttccc tcagttcatt tcttcttctt tgatctacgt ttgtttgtct
     2521 ttttaaatat tgccttgtaa cttgctcaga ggacaaggaa gatatgtccc tgtttcttct
     2581 catagctctc aagaatagta gcataattgg cttttatgcc agggtgacag gggaagaata
     2641 tattttacat ataaattctg tttgacatag gattcttata ataatttgtc agtagtttaa
     2701 ggttgcaaac aaatgtcttt gtaaataagc ctgcagtatc tggtattttt gctctacagt
     2761 tatgttgatg gttcttccat attcccacag ctcctgggca atatgatcgt gattgtgctg
     2821 ggccaccacc tgggcaagga tttcaccccc gctgcacagg ctgccttcca gaaggtggtg
     2881 gctggagtgg ccactgccct ggctcacaag taccacggta cctacccata cgatgttccg
     2941 gattacgctt agatcacggg aaagatcgcc gtgtaagcgg ccgcctagtt gccagccatc
     3001 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     3061 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     3121 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     3181 ggatgcggtg ggctctatgg ccggcttgtc gacgacggcg gactccgtcg tcaggatcat
     3241 ccgcgctagc caccatggtg agcaagggcg aggaggataa catggccatc atcaaggagt
     3301 tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg
     3361 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
     3421 gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg
     3481 cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct
     3541 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact
     3601 cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct
     3661 ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt
     3721 accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg
     3781 gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg
     3841 gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg
     3901 tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca
     3961 aggagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc ggccctagga
     4021 tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac
     4081 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc
     4141 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca
     4201 tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga
     4261 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt
     4321 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc
     4381 ccgcgtggtt cctggccacc gtcggcgtca gccccgacca ccagggcaag ggtctgggca
     4441 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg
     4501 agacatccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg
     4561 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa
     4621 tcacgccatc agagagatcc tcgcggccgc ggatccagac atgataagat acattgatga
     4681 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     4741 tgctattgct ttatttgtaa ccattataag ctgcaataaa ctctagagag tgattatcaa
     4801 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     4861 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     4921 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     4981 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgggac ccacgctcac
     5041 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5101 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5161 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5221 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5281 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5341 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5401 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5461 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     5521 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     5581 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     5641 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     5701 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     5761 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     5821 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgtca
     5881 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5941 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     6001 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     6061 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     6121 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     6181 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6241 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6301 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6361 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6421 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6481 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6541 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     6601 tgttctttcc tgcgttatcc cctgattctg tggataaccg t
//
