LOCUS       sequence-497342-        7521 bp    DNA     circular SYN 14-JAN-2026
DEFINITION  Expresses optogenetic LRP6 in mammalian cells; no fluorescent 
            proteins. Plasmid contains a puromycin selection marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7521)
  AUTHORS   Rosen SJ, Witteveen O, Baxter N, Lach RS, Hopkins E, Bauer M, Wilson
            MZ
  TITLE     Anti-resonance in developmental signaling regulates cell fate 
            decisions.
  JOURNAL   bioRxiv [Preprint]. 2025 May 23:2025.02.04.636331. doi: 
            10.1101/2025.02.04.636331.
  PUBMED    39990305
REFERENCE   2  (bases 1 to 7521)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 14, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2025 May 23:2025.02.04.636331. doi: 
            10.1101/2025.02.04.636331."
COMMENT     Sequence Label: pPig-hEF1a-Cry2PHR-LRP6c-Puro-Blind
FEATURES             Location/Qualifiers
     source          1..7521
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      168..756
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     657..676
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     910..927
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1044..1065
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1080..1085,1086..1103,1104..1110)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1118..1134
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     1123..1145
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1142..1158
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1142..1158
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   1193..1227
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1710..2896
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1941..2887
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2844..2864
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    2969..2993
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3003..4496
                     /codon_start=1
                     /product="N-terminal photolyase homology region of 
                     Arabidopsis thaliana cryptochrome 2"
                     /label=CRY2PHR
                     /note="binds to CIB1 upon exposure to blue light (Kennedy 
                     et al., 2010)"
                     /translation="MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQ
                     FYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLY
                     DPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSI
                     ESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLN
                     EFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGE
                     ESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPL
                     VDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQY
                     ISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKA
                     SGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAA"
     CDS             5217..5273
                     /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             5274..5873
                     /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(5274..5293)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5770..5790
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     repeat_region   6114..6176
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6288..6307)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6407..6429
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6467..6485)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6553..6657
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6658..6726,6727..7518)
                     /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     complement(6876..6895)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
       61 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
      121 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
      181 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
      241 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
      301 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
      361 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
      421 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
      481 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
      541 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
      601 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
      661 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
      721 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
      781 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
      841 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
      901 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
      961 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     1021 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     1081 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     1141 acaggaaaca gctatgacca tgattacgcc ggggcgcgcc attctagatt aaccctagaa
     1201 agatagtctg cgtaaaattg acgcatgcat tcttgaaata ttgctctctc tttctaaata
     1261 gcgcgaatcc gtcgctgtgc atttaggaca tctcagtcgc cgcttggagc tcccgtgagg
     1321 cgtgcttgtc aatgcggtaa gtgtcactga ttttgaacta taacgaccgc gtgagtcaaa
     1381 atgacgcatg attatctttt acgtgacttt taagatttaa ctcatacgat aattatattg
     1441 ttatttcatg ttctacttac gtgataactt attatatata tattttcttg ttatagatat
     1501 caactagaat gctagcatgg gcccagatct ccgaggggac ccgacaggcc cggtctgctt
     1561 tttgcttgta ctgtcgaccc tgtggaatga ccgtcgacct cgacggatcg ggagatctcg
     1621 accgagcttt gcaaagatgg ataaagtttt aaacagagag gaatctttgc agctaatgga
     1681 ccttctaggt cttgaaagga gtgggaattg gctccggtgc ccgtcagtgg gcagagcgca
     1741 catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga
     1801 gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg
     1861 agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg
     1921 ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta
     1981 cgggttatgg cccttgcgtg ccttgaatta cttccacctg gctgcagtac gtgattcttg
     2041 atcccgagct tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc
     2101 cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct
     2161 ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt
     2221 gatgacctgc tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc
     2281 gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc
     2341 cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg
     2401 tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg
     2461 ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt
     2521 cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt
     2581 gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc
     2641 acggagtacc gggcgccgtc caggcacctc gattagttct cgatcgagct tttggagtac
     2701 gtcgtcttta ggttgggggg aggggtttta tgcgatggag tttccccaca ctgagtgggt
     2761 ggagactgaa gttaggccag cttggcactt gatgtaattc tccttggaat ttgccctttt
     2821 tgagtttgga tcttggttca ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc
     2881 atttcaggtg tcgtgaggaa ttagcttgac caagcttggt accgagctcg gatccactag
     2941 taacggccgc cagtgtgctg gaattatcac aagtttgtac aaaaaagcag gctgctatga
     3001 ccatgaagat ggacaaaaag actatagttt ggtttagaag agacctaagg attgaggata
     3061 atcctgcatt agcagcagct gctcacgaag gatctgtttt tcctgtcttc atttggtgtc
     3121 ctgaagaaga aggacagttt tatcctggaa gagcttcaag atggtggatg aaacaatcac
     3181 ttgctcactt atctcaatcc ttgaaggctc ttggatctga cctcacttta atcaaaaccc
     3241 acaacacgat ttcagcgatc ttggattgta tccgcgttac cggtgctaca aaagtcgtct
     3301 ttaaccacct ctatgatcct gtttcgttag ttcgggacca taccgtaaag gagaagctgg
     3361 tggaacgtgg gatctctgtg caaagctaca atggagatct attgtatgaa ccgtgggaga
     3421 tatactgcga aaagggcaaa ccttttacga gtttcaattc ttactggaag aaatgcttag
     3481 atatgtcgat tgaatccgtt atgcttcctc ctccttggcg gttgatgcca ataactgcag
     3541 cggctgaagc gatttgggcg tgttcgattg aagaactagg gctggagaat gaggccgaga
     3601 aaccgagcaa tgcgttgtta actagagctt ggtctccagg atggagcaat gctgataagt
     3661 tactaaatga gttcatcgag aagcagttga tagattatgc aaagaacagc aagaaagttg
     3721 ttgggaattc tacttcacta ctttctccgt atctccattt cggggaaata agcgtcagac
     3781 acgttttcca gtgtgcccgg atgaaacaaa ttatatgggc aagagataag aacagtgaag
     3841 gagaagaaag tgcagatctt tttcttaggg gaatcggttt aagagagtat tctcggtata
     3901 tatgtttcaa cttcccgttt actcacgagc aatcgttgtt gagtcatctt cggtttttcc
     3961 cttgggatgc tgatgttgat aagttcaagg cctggagaca aggcaggacc ggttatccgt
     4021 tggtggatgc cggaatgaga gagctttggg ctaccggatg gatgcataac agaataagag
     4081 tgattgtttc aagctttgct gtgaagtttc ttctccttcc atggaaatgg ggaatgaagt
     4141 atttctggga tacacttttg gatgctgatt tggaatgtga catccttggc tggcagtata
     4201 tctctgggag tatccccgat ggccacgagc ttgatcgctt ggacaatccc gcgttacaag
     4261 gcgccaaata tgacccagaa ggtgagtaca taaggcaatg gcttcccgag cttgcgagat
     4321 tgccaactga atggatccat catccatggg acgctccatt aaccgtactc aaagcttctg
     4381 gtgtggaact cggaacaaac tatgcgaaac ccattgtaga catcgacaca gctcgtgagc
     4441 tactagctaa agctatttca agaacccgtg aagcacagat catgatcgga gcagcagccc
     4501 gggatccacc ggtcgccacc gatggtggag gaggtagtgg aggaggagga tcaaggatgt
     4561 tgtgtccacg tatgaaggga gatggggaaa ctatgactaa tgactatgta gttcatggac
     4621 cagcttctgt gcctcttggt tatgtgccac acccaagttc tttgtcagga tctcttccag
     4681 gaatgtctcg aggtaaatca atgatcagct ccctcagtat catgggggga agcagtggac
     4741 ccccctatga ccgagcccat gttacaggag catcatcaag tagttcttca agcaccaaag
     4801 gcacttactt ccctgcaatt ttgaaccctc caccatcccc agccacagag cgatcacatt
     4861 acactatgga atttggatat tcttcaaaca gtccttccac tcataggtca tacagctaca
     4921 ggccatatag ctaccggcac tttgcacccc ccaccacacc ctgcagcaca gatgtttgtg
     4981 acagtgacta tgctcctagt cggagaatga cctcagtggc aacagccaag ggctatacca
     5041 gtgacttgaa ctatgattca gaacctgtgc ccccacctcc cacaccccga agccaatact
     5101 tgtcagcaga ggagaactat gaaagctgcc caccttctcc atacacagag aggagctatt
     5161 ctcatcacct ctacccaccg ccaccctctc cctgtacaga ctcctccgga agcggagcta
     5221 ctaacttcag cctgctgaag caggctggag acgtggagga gaaccctgga cctatgaccg
     5281 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc
     5341 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca
     5401 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
     5461 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
     5521 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
     5581 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     5641 ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg
     5701 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct
     5761 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     5821 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgacgcccgc
     5881 cccacgaccc gcagcgcccg accgaaagga gcgcacgacc ccatgcatcg atactagtta
     5941 aaagttttgt tactttatag aagaaatttt gagtttttgt ttttttttaa taaataaata
     6001 aacataaata aattgtttgt tgaatttatt attagtatgt aagtgtaaat ataataaaac
     6061 ttaatatcta ttcaaattaa taaataaacc tcgatataca gaccgataaa acacatgcgt
     6121 caattttacg catgattatc tttaacgtac gtcacaatat gattatcttt ctagggttaa
     6181 tctagtatac gcgtatgcgg ccgcttaatt aatccatggc gcctgatgcg gtattttctc
     6241 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct
     6301 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     6361 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     6421 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     6481 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     6541 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     6601 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     6661 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     6721 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     6781 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     6841 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     6901 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     6961 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     7021 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     7081 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     7141 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     7201 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     7261 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     7321 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     7381 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     7441 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     7501 ctgattaagc attggtaact g
//
