LOCUS       sequence-455144-        6642 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1 attB_GA mCherry-T2A-puro, Dox-inducible beta-globin 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 6642)
  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 6642)
  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: pHPHS927
FEATURES             Location/Qualifiers
     source          1..6642
                     /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             2924..2950
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    3204..3241
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     regulatory      3250..3259
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3256..3963
                     /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(3401..3419)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3669..3688
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3964..4017
                     /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             4021..4620
                     /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(4021..4040)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4517..4537
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4677..4696)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4731..4750
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(4887..5678,5679..5747))
                     /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     5510..5529
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5748..5852)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5955..6543
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6444..6463
                     /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 ctctctgcct attggtctat tttcccaccc ttaggctgct
     1921 ggtggtctac ccttggaccc agaggttctt tgagtccttt ggggatctgt ccactcctga
     1981 tgctgttatg ggcaacccta aggtgaaggc tcatggcaag aaagtgctcg gtgcctttag
     2041 tgatggcctg gctcacctgg acaacctcaa gggcaccttt gccacactga gtgagctgca
     2101 ctgtgacaag ctgcacgtgg atcctgagaa cttcagggtg agtctgatgg gcacctcctg
     2161 ggtttccttc ccctggctat tctgctcaac cttcctatca gaaaaaaagg ggaagcgatt
     2221 ctagggagca gtctccatga ctgtgtgtgg agtgttgaca agagttcgga tattttattc
     2281 tctactcaga attgctgctc cccctcactc tgttctgtgt tgtcatttcc tctttctttg
     2341 gtaagctttt aatttccagt tgcattttac taaattaatt aagctggtta tttacttccc
     2401 atcctgatat cagcttcccc tcctcctttc ctcccagtcc ttctctctct cctctctctt
     2461 tctctaatcc tttcctttcc ctcagttcat ttcttcttct ttgatctacg tttgtttgtc
     2521 tttttaaata ttgccttgta acttgctcag aggacaagga agatatgtcc ctgtttcttc
     2581 tcatagctct caagaatagt agcataattg gcttttatgc cagggtgaca ggggaagaat
     2641 atattttaca tataaattct gtttgacata ggattcttat aataatttgt cagtagttta
     2701 aggttgcaaa caaatgtctt tgtaaataag cctgcagtat ctggtatttt tgctctacag
     2761 ttatgttgat ggttcttcca tattcccaca gctcctgggc aatatgatcg tgattgtgct
     2821 gggccaccac ctgggcaagg atttcacccc cgctgcacag gctgccttcc agaaggtggt
     2881 ggctggagtg gccactgccc tggctcacaa gtaccacggt acctacccat acgatgttcc
     2941 ggattacgct tagacagtgg gaaagatcgc cgtgtaagcg gccgcctagt tgccagccat
     3001 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     3061 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     3121 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     3181 gggatgcggt gggctctatg gccggcttgt cgacgacggc ggactccgtc gtcaggatca
     3241 tccgcgctag ccaccatggt gagcaagggc gaggaggata acatggccat catcaaggag
     3301 ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag
     3361 ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag
     3421 ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag
     3481 gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc
     3541 ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac
     3601 tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc
     3661 tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg
     3721 taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc
     3781 ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc
     3841 ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc
     3901 gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac
     3961 aaggagggca gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccctagg
     4021 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     4081 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
     4141 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     4201 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     4261 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     4321 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     4381 cccgcgtggt tcctggccac cgtcggcgtc agccccgacc accagggcaa gggtctgggc
     4441 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     4501 gagacatccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     4561 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga
     4621 acagtgccat cagagagatc ctcgcggccg cggatccaga catgataaga tacattgatg
     4681 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     4741 atgctattgc tttatttgta accattataa gctgcaataa actctagaga gtgattatca
     4801 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4861 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4921 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4981 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcggga cccacgctca
     5041 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5101 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5161 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5221 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5281 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5341 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5401 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5461 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5521 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5581 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5641 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     5701 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     5761 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5821 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgtc
     5881 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     5941 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     6001 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     6061 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     6121 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     6181 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     6241 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     6301 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     6361 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     6421 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     6481 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     6541 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     6601 atgttctttc ctgcgttatc ccctgattct gtggataacc gt
//
