LOCUS       sequence-483142-        7496 bp    DNA     circular SYN 13-AUG-2025
DEFINITION  C_terminal segment of a blue_light_controlled split PS_intein tTA 
            gene_expression system, co_expressing mCherry under an 
            NF_KB_inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7496)
  TITLE     Photoswitchable intein for light control of covalent protein binding
            and cleavage
REFERENCE   2  (bases 1 to 7496)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 13, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pNF-KB-Split PS Intein-C tTA
FEATURES             Location/Qualifiers
     source          1..7496
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    7..98
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     47..66
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        214..245
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     CDS             290..1225
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             1832..2281
                     /codon_start=1
                     /product="""positive Magnet"" variant of an N-terminally 
                     truncated Vivid (VVD) blue light photoreceptor from 
                     Neurospora crassa (Kawano et al., 2015)"
                     /label=pMag
                     /note="rapidly heterodimerizes with nMagHigh1 upon blue 
                     light irradiation to form a complex with slow switch-off 
                     kinetics"
                     /translation="HTLYAPGGYDIMGYLIQIMNRPNPQVELGPVDTSCALILCDLKQK
                     DTPIVYASEAFLYMTGYSNAEVLGRNCRFLQSPDGMVKPKSTRKYVDSNTINTMRKAID
                     RNAEVQVEVVNFKKNGQRFVNFLTMIPVRDETGEYRYSMGFQCETE"
     CDS             join(2369..2986,2987..3370)
                     /codon_start=1
                     /product="tetracycline-controlled transactivator, 
                     comprising a fusion of the tetracycline repressor TetR with
                     the C-terminal activation domain of herpes simplex virus 
                     VP16"
                     /label=tTA
                     /note="In the Tet-Off(R) system, tTA binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="SRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVK
                     NKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRP
                     TEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTD
                     SMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIE
                     GLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHAD
                     ALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     primer_bind     complement(2439..2458)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             3386..3439
                     /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             3440..4150
                     /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(3585..3603)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3853..3872
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4273..4394
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4310..4329)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4364..4383
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4401..4856)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4538..4559)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4750..4769
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4883..4987
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4989..5346
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4989..5180
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5012..5032)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5197..5332
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5381..6175
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             5381..6175
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5435..5454)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6045..6064
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6363..6382)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6407..6454
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6783..7371
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7272..7291
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agtactaaca tacgctctcc atcaaaacaa aacgaaacaa aacaaactag caaaataggc
       61 tgtccccagt gcaagtgcag gtgccagaac atttctctgg cctaactggc cggtacctga
      121 gctcgctagc gggaatttcc ggggactttc cgggaatttc cggggacttt ccgggaattt
      181 ccagatctgg cctcggcggc caagcttaga cactagaggg tatataatgg aagctcgact
      241 tccagcttgg caatccggta ctgttggtaa agccaccgct agcaccatgg ctggagacat
      301 gagagctgcc aacctttggc caagcccgct catgatcaaa cgctctaaga agaacagcct
      361 ggccttgtcc ctgacggccg accagatggt cagtgccttg ttggatgctg agccccccat
      421 actctattcc gagtatgatc ctaccagacc cttcagtgaa gcttcgatga tgggcttact
      481 gaccaacctg gcagacaggg agctggttca catgatcaac tgggcgaaga gggtgccagg
      541 ctttgtggat ttgaccctcc atgatcaggt ccaccttcta gaatgtgcct ggctagagat
      601 cctgatgatt ggtctcgtct ggcgctccat ggagcaccca gtgaagctac tgtttgctcc
      661 taacttgctc ttggacagga accagggaaa atgtgtagag ggcatggtgg agatcttcga
      721 catgctgctg gctacatcat ctcggttccg catgatgaat ctgcagggag aggagtttgt
      781 gtgcctcaaa tctattattt tgcttaattc tggagtgtac acatttctgt ccagcaccct
      841 gaagtctctg gaagagaagg accatatcca ccgagtcctg gacaagatca cagacacttt
      901 gatccacctg atggccaagg caggcctgac cctgcagcag cagcaccagc ggctggccca
      961 gctcctcctc atcctctccc acatcaggca catgagtaac aaaggcatgg agcatctgta
     1021 cagcatgaag tgcaagaacg tggtgcccct ctatgacctg ctgctggagg cggcggacgc
     1081 ccaccgccta catgcgccca ctagccgtgg aggggcatcc gtggaggaga cggaccaaag
     1141 ccacttggcc actgcgggct ctacttcatc gcattccttg caaaagtatt acatcacggg
     1201 ggaggcagag ggtttccctg ccacagctgg gggtggtgga tcaaccggtg aaatgatgct
     1261 gaaaaaaatc ctgaaaatcg aggaactgga tgaacgcggt accggcagcg gaggtcatac
     1321 cttgtatgcg ccggggggtt atgacatcat gggttacctc atacagatca tgaataggcc
     1381 gaacccacaa gtggaactcg gacccgtcga tacctcctgc gctctcattc tgtgtgacct
     1441 taagcagaag gataccccta tcgtgtacgc ctccgaggca tttctgtaca tgacagggta
     1501 ctcgaacgcc gaagtgctgg gacggaactg ccgcttcctg caaagcccgg atggaatggt
     1561 gaagcctaag tcaacccgca aatacgtgga ctccaacact atcaacacca tgcgcaaggc
     1621 cattgaccgc aatgctgagg tgcaagtgga agtggtgaac ttcaagaaga atggacagcg
     1681 cttcgtcaac ttcctgacta tgatccccgt gcgcgacgag accggcgaat atcggtacag
     1741 catggggttt cagtgtgaga cagagggcgg ctcaggaggc agcggcggtt ctggaggttc
     1801 cggtggcggc tcaggaggta gcggagggtc tcacactctt tacgcccctg gaggatacga
     1861 cattatggga tatttgattc agattatgaa ccgcccaaac cctcaggtcg aactggggcc
     1921 tgtggacacg tcatgtgccc tgatcctgtg cgatctgaag caaaaggaca ctccgattgt
     1981 ctacgcctcg gaagccttct tgtatatgac cggatacagc aatgcagagg tgctcggtag
     2041 gaactgcaga ttcctgcagt cccccgacgg gatggtgaaa ccaaagtcca ctcgcaaata
     2101 tgtggactcg aacacgatca atacaatgcg gaaggccatc gaccggaacg ccgaggtcca
     2161 ggtggaggtg gtcaacttta agaagaacgg ccagcggttc gtgaactttc tcaccatgat
     2221 tccggtccgg gatgaaaccg gagagtacag atactccatg ggattccagt gcgaaaccga
     2281 agggtcagga tccgaactga ttgatattga agttagcggt aaccacctgt tctatgccaa
     2341 tgatattctg acccataata gctcgagctc tagattagat aaaagtaaag tgattaacag
     2401 cgcattagag ctgcttaatg aggtcggaat cgaaggttta acaacccgta aactcgccca
     2461 gaagctaggt gtagagcagc ctacattgta ttggcatgta aaaaataagc gggctttgct
     2521 cgacgcctta gccattgaga tgttagatag gcaccatact cacttttgcc ctttagaagg
     2581 ggaaagctgg caagattttt tacgtaataa cgctaaaagt tttagatgtg ctttactaag
     2641 tcatcgcgat ggagcaaaag tacatttagg tacacggcct acagaaaaac agtatgaaac
     2701 tctcgaaaat caattagcct ttttatgcca acaaggtttt tcactagaga atgcattata
     2761 tgcactcagc gctgtggggc attttacttt aggttgcgta ttggaagatc aagagcatca
     2821 agtcgctaaa gaagaaaggg aaacacctac tactgatagt atgccgccat tattacgaca
     2881 agctatcgaa ttatttgatc accaaggtgc agagccagcc ttcttattcg gccttgaatt
     2941 gatcatatgc ggattagaaa aacaacttaa atgtgaaagt gggtccgcgt acagccgcgc
     3001 gcgtacgaaa aacaattacg ggtctaccat cgagggcctg ctcgatctcc cggacgacga
     3061 cgcccccgaa gaggcggggc tggcggctcc gcgcctgtcc tttctccccg cgggacacac
     3121 gcgcagactg tctacggccc ccccgaccga tgtcagcctg ggggacgagc tccacttaga
     3181 cggcgaggac gtggcgatgg cgcatgccga cgcgctagac gatttcgatc tggacatgtt
     3241 gggggacggg gattccccgg gtccgggatt taccccccac gactccgccc cctacggcgc
     3301 tctggatatg gccgacttcg agtttgagca gatgtttacc gatgcccttg gaattgacga
     3361 gtacggtggg gaattcggta gcggcgaggg cagaggaagt ctgctaacat gcggtgatgt
     3421 cgaggagaat cctggcccaa tggtgagcaa gggcgaggag gataacatgg ccatcatcaa
     3481 ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat
     3541 cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac
     3601 caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc
     3661 caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga
     3721 gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca
     3781 ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt
     3841 cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg
     3901 gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga
     3961 cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct
     4021 gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac
     4081 catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct
     4141 gtacaagtaa agcggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt
     4201 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     4261 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     4321 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     4381 atcaatgtat cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     4441 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     4501 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     4561 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     4621 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     4681 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     4741 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     4801 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg
     4861 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4921 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     4981 gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     5041 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     5101 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     5161 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     5221 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     5281 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     5341 tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     5401 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     5461 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     5521 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct
     5581 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     5641 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     5701 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     5761 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     5821 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     5881 agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac
     5941 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     6001 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     6061 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     6121 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     6181 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     6241 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     6301 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta
     6361 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     6421 cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc
     6481 tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc
     6541 ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg
     6601 gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt
     6661 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6721 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6781 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6841 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6901 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6961 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     7021 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     7081 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     7141 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     7201 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     7261 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     7321 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     7381 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     7441 gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcattagtt attaat
//
