LOCUS       sequence-444708-        6590 bp    DNA     circular SYN 15-APR-2025
DEFINITION  PhoBIT2 component; sspB2 (sspB mutant A56F) fused to mCh-CRY2PHR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6590)
  TITLE     Engineering of a bacterial two component system for optogenetic 
            control of biological processes
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6590)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: mCh-CRY2-sspB2
FEATURES             Location/Qualifiers
     source          1..6590
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             603..1310
                     /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(748..766)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1016..1035
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             complement(2863..2880)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2913..3242
                     /codon_start=1
                     /product="SspB from Haemophilus influenzae, with the 
                     mutations Y11K and A15E to disrupt the natural dimerization
                     of the protein"
                     /label=SspB nano
                     /note="dimerizes with iLID at high affinity to create iLID 
                     nano (Guntas et al., 2015)"
                     /translation="SSPKRPKLLREYYDWLVDNSFTPYLVVDATYLGVNVPVEYVKDGQ
                     IVLNLSASFTGNLQLTNDFIQFNARFKGVSRELYIPMGAALAIYARENGDGVMFEPEEI
                     YDELNI"
     polyA_signal    3378..3499
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3415..3434)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3469..3488
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3506..3961)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3643..3664)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3855..3874
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3988..4092
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4094..4451
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4094..4285
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4117..4137)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4302..4437
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4486..5280
                     /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             4486..5280
                     /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(4540..4559)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5150..5169
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5468..5487)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5512..5559
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5888..6476
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6377..6396
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcggtg
      601 gaatggtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca
      661 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
      721 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
      781 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc
      841 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc
      901 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg
      961 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg
     1021 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg
     1081 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg
     1141 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg
     1201 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg
     1261 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag ggtagcggac
     1321 caccggtcgc caccatgaag atggacaaaa agaccatcgt ctggtttcgg agagatttga
     1381 gaatagaaga taatcccgcg ctcgccgccg cggcccacga gggttccgtc ttccccgttt
     1441 tcatttggtg tcctgaagaa gaaggccagt tttatcccgg aagggcctct aggtggtgga
     1501 tgaagcaaag tctggcccat cttagccagt cactgaaagc actgggcagt gatcttaccc
     1561 tgatcaagac acacaatacc atctctgcca tcctcgactg catcagggtg accggcgcaa
     1621 cgaaagtcgt gtttaaccac ctgtacgatc cagttagtct ggtgcgcgac cacactgtga
     1681 aggagaagct ggtggaacgg gggatcagtg tgcagagcta caacggggac cttctgtacg
     1741 agccatggga gatctattgc gagaaaggga aaccgttcac ctccttcaac agttactgga
     1801 agaaatgttt ggatatgtca atagagtccg ttatgttgcc ccctccctgg agactgatgc
     1861 cgattactgc tgctgcagag gccatctggg cctgctccat cgaggaactc ggtctggaaa
     1921 atgaagcaga aaagccaagc aatgcacttc tcactagagc ctggagcccc ggctggtcta
     1981 atgccgacaa gctgcttaac gagttcatcg aaaaacaact gattgactac gcgaagaact
     2041 ccaagaaagt ggtaggtaac tcaactagct tgctctctcc atatctccat tttggcgaga
     2101 tttctgtccg ccatgtattt cagtgcgctc ggatgaaaca gattatctgg gctcgcgata
     2161 aaaacagcga aggcgaagaa agcgccgatc tgttcctgcg agggatcgga cttcgggaat
     2221 actcccggta tatatgtttc aactttccat tcacacacga gcagagtctg ttgtcccacc
     2281 tcaggttctt cccctgggac gccgatgtcg acaaattcaa ggcatggaga cagggaagga
     2341 cagggtaccc actcgtggat gctggcatga gagagctctg ggctacaggc tggatgcaca
     2401 accgcatccg ggtaatcgtg tcctcatttg ctgtcaagtt tctgctcctg ccttggaaat
     2461 ggggaatgaa gtacttttgg gatacccttc tcgacgccga cttggagtgt gacattctgg
     2521 gatggcaata tattagcggg tcaattcctg acggccatga gttggacagg ttggacaatc
     2581 cggccttgca gggagctaag tatgatcccg aaggagagta tattcgacag tggctccccg
     2641 agctggcccg acttcctacg gagtggattc accatccttg ggacgcacca ctgacagtgc
     2701 tcaaggcaag cggggtggag ctgggcacca attacgctaa gcctatagtt gatatagata
     2761 cagcacgcga gctgctggct aaagcgatct ctcgcactcg ggaggcgcag attatgatcg
     2821 gtgctgccgt gtacagtggg ggaggcggag ggggtggttc aggtggtggt ggtggtggtg
     2881 gttcgggtgg tggcggaggg ggaggctccg gaagctcccc gaaacgccct aagctgctgc
     2941 gtgaatatta cgattggctg gttgataaca gctttacccc atatctggtg gtggatgcca
     3001 catacctggg cgtgaacgtg cccgtggagt atgtgaaaga cggtcagatc gtgctgaatc
     3061 tgtctgcaag tttcaccggc aacctgcaac tgacaaatga ttttatccag ttcaacgccc
     3121 gctttaaggg cgtgtctcgt gaactgtata tcccgatggg tgccgctctg gccatttacg
     3181 ctcgcgagaa cggcgatggt gtgatgttcg aaccagaaga aatctatgac gagctgaata
     3241 ttggttaggg atccaccgga tctagataac tgatcataat cagccatacc acatttgtag
     3301 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     3361 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     3421 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     3481 aactcatcaa tgtatcttaa cgcgtaaatt gtaagcgtta atattttgtt aaaattcgcg
     3541 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     3601 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     3661 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     3721 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     3781 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     3841 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     3901 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     3961 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4021 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     4081 aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg
     4141 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     4201 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     4261 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     4321 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     4381 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     4441 gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     4501 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     4561 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     4621 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg
     4681 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     4741 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     4801 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     4861 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     4921 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     4981 gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc
     5041 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     5101 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     5161 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     5221 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     5281 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     5341 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     5401 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga
     5461 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
     5521 aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc
     5581 agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt
     5641 cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg
     5701 tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag attgatttaa
     5761 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     5821 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     5881 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     5941 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     6001 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     6061 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     6121 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     6181 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     6241 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     6301 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     6361 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6421 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     6481 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     6541 ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat
//
