LOCUS       sequence-460758-        7256 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid mediates puromycin resistance in the dark, resulting in
            puro mediated cell death upon illumination with blue light.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7256)
  AUTHORS   Wolf B, Shehu P, Brenker L, von Bachmann A, Kroell A-S, Southern N, 
            Holderbach S, Eigenmann J, Aschenbrenner S, Mathony J, Niopek D
  TITLE     Rational engineering of allosteric protein switches by in silico 
            prediction of domain insertion sites
  JOURNAL   bioRxiv 2024.12.04.626757
REFERENCE   2  (bases 1 to 7256)
  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"; journalName: "bioRxiv 
            2024.12.04.626757"
COMMENT     Sequence Label: PAC_E83_LOV_eGFP
FEATURES             Location/Qualifiers
     source          1..7256
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             380..433
                     /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             join(434..436,437..439,440..1153)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(434..436,437..439,440..1150)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(479..500)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(740..759)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1087..1108
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1225..1242)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1231..1455
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1501..1929
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1588..1607)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1798..1819
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1938..1958)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1943..2272
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2123..2258
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2185..2204
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2339..3133
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2393..2412)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3003..3022
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3307..3428
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3344..3363)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3398..3417
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3477..3493)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3477..3493)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3490..3512)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3501..3517
                     /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)."
     promoter        complement(join(3525..3531,3532..3549,3550..3555))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3570..3591
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3708..3725)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3879..4467)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3959..3978)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4638..5429,5430..5498))
                     /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     5261..5280
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5499..5603)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5678..5697)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5869..6248
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6249..6452
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6403..6423
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6497..6516
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6497..6515
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gagcgccggt gctgtgttcg ccgagatcgg cccgcgcatg gctgaactct ccggaagtag
       61 acttgcagcc cagcaacaga tggaggggct tctcgcacca caccgcccca aagaaccagc
      121 ttggttcctt gctactgtgg gcgtgtcccc ggatcaccag gggaagggac ttggaagtgc
      181 agtagtgctt cctggggtcg aggctgcgga acgcgctggt gtcccagctt ttttggaaac
      241 ttccgccccg cgcaacctgc cattctatga gagactcggc tttactgtta ctgcagatgt
      301 cgaagtacct gagggaccta ggacttggtg tatgacccgc aagcctgggg ctggctccac
      361 agggagcaga ggaagcggag agggcagggg aagtcttcta acatgcgggg acgtggagga
      421 aaatcccggc cccatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
      481 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
      541 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
      601 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
      661 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
      721 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
      781 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
      841 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
      901 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
      961 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     1021 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     1081 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     1141 gctgtacaag tgagcttgaa ttccaccaca ctggactagt ggatccgagc tcggtaccaa
     1201 gcttaagttt aaaccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     1261 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     1321 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     1381 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     1441 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
     1501 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     1561 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     1621 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     1681 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     1741 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     1801 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     1861 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     1921 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     1981 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     2041 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     2101 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     2161 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     2221 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     2281 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat
     2341 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     2401 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     2461 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     2521 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     2581 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     2641 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     2701 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     2761 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     2821 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     2881 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     2941 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     3001 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     3061 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     3121 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     3181 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     3241 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     3301 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3361 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3421 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     3481 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3541 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     3601 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3661 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     3721 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3781 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3841 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3901 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3961 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4021 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4081 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4141 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4201 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4261 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4321 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4381 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4441 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4501 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4561 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4621 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4681 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4741 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4801 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4861 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4921 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     4981 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5041 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5101 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5161 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5221 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5281 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5341 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5401 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5461 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5521 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5581 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     5641 tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat
     5701 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     5761 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     5821 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
     5881 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     5941 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     6001 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     6061 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     6121 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     6181 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     6241 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
     6301 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
     6361 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
     6421 gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa cccactgctt
     6481 actggcttat cgaaattaat acgactcact atagggagac ccaagctggc tagcgtttaa
     6541 acgggccctc tagactcgag ggaggccacc atgactgagt ataaaccaac agttaggttg
     6601 gcaacaaggg acgatgtccc gcgagcggtt agaacacttg ctgccgcttt tgcggattat
     6661 cccgcgaccc ggcacaccgt agacccggac agacatatag aacgagtcac ggaacttcaa
     6721 gagttgtttc ttacaagagt agggctcgac atcgggaagg tctgggttgc tgatgatgga
     6781 gctgcagtag ctgtttggac aacccctgaa tcagtggaat ctggtcttgc tacaactttg
     6841 gagcggattg agaagaattt cgtcataact gatcccaggt tgcctgataa tcctataata
     6901 ttcgcctccg atagttttct tcaacttacg gagtactcca gagaggagat actgggaagg
     6961 aattgcagat tcctccaagg tccagaaact gatagagcta ctgtgaggaa gatacgggat
     7021 gccattgaca atcaaactga agtgacagtg caacttatca attacaccaa gtcagggaaa
     7081 aaattctgga acctgttcca tttgcaaccc atgagggacc aaaagggcga tgtgcaatat
     7141 ttcataggcg ttcaacttga tggtactgaa cacgtcagag atgcggcgga gcgagaagga
     7201 gtgatgctga ttaagaaaac agcagagaat atcgacgaag cggcgaaaga gttggg
//
