LOCUS       sequence-444732-        6122 bp    DNA     circular SYN 15-APR-2025
DEFINITION  PhoBIT2 component; ssrA2 tag (ssrA mutant A2C) inserted into 
            mCerulean-tagged STIM1 cytoplasmic domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6122)
  TITLE     Engineering of a bacterial two component system for optogenetic 
            control of biological processes
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6122)
  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            "Unpublished"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: STIM1ct-ssrA2-mCerulean
FEATURES             Location/Qualifiers
     source          1..6122
                     /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"
     regulatory      2062..2071
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2068..2070,2071..2073,2074..2787)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2113..2134)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2374..2393)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2721..2742
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2910..3031
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2947..2966)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3001..3020
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3038..3493)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3175..3196)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3387..3406
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3520..3624
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3626..3983
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3626..3817
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3649..3669)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3834..3969
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4018..4812
                     /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             4018..4812
                     /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(4072..4091)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4682..4701
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5000..5019)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5044..5091
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5420..6008
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5909..5928
                     /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 gctagcgcta
      601 ccggactcag atctcgagat gcagaaccgt tactccaagg agcacatgaa gaagatgatg
      661 aaggacttgg aggggttaca ccgagctgag cagagtctgc atgaccttca ggaaaggctg
      721 cacaaggccc aggaggagca ccgcacagtg gaggtggaga aggtccatct ggaaaagaag
      781 ctgcgcgatg agatcaacct tgctaagcag gaagcccagc ggctgaagga gctgcgggag
      841 ggtactgaga atgagcggag ccgccaaaaa tatgctgagg aggagttgga gcaggttcgg
      901 gaggccttga ggaaagcaga gaaggagcta gaatctcaca gctcatggta tgctccagag
      961 gcccttcaga agtggctgca gctgacacat gaggtggagg tgcaatatta caacatcaag
     1021 aagcaaaatg ctgagaagca gctgctggtg gccaaggagg gggctgagaa gataaaaaag
     1081 aagagaaaca cactctttgg caccttccac gtggcccaca gctcttccct ggatgatgta
     1141 gatcataaaa ttctaacagc taagcaagca ctgagcgagg tgacagcagc attgcgggag
     1201 cgcctgcacc gctggcaaca gatcgagatc ctctgtggct tccagattgt caacaaccct
     1261 ggcatccact cactggtggc tgccctcaac atagacccca gctggatggg cagtacacgc
     1321 cccaaccctg ctcacttcat catgactgac gacgtggatg acatggatga ggagattgtg
     1381 tctcccttgt ccatgcagtc ccctagcctg cagagcagtg ttcggcagcg cctgacggag
     1441 ccacagcatg gcctgggatc tcagagggat ttgacccatt ccgattcgga gtcctccctc
     1501 cacatgagtg accgccagcg tgtggccccc aaacctcctc agatgagccg tgctgcagac
     1561 gaggctctca atgccatgac ttccaatggc agccaccggc tgatcgaggg ggtccaccca
     1621 gggtctctgg tggagaaact gcctgacagc cctgccctgg ccaagaaggc attactggcg
     1681 ctgaaccatg ggctggacaa ggcccacagc ctgatggagc tgagcccctc agccccacct
     1741 ggtggctctc cacatttgga ttcttcccgt tctcacagcc ccagctcccc agacccagac
     1801 acaccatctc cagttgggga cagccgagcc ctgcaagcca gccgaaacac acgcattccc
     1861 cacctggctg gcaagaaggc tgtggctgag gaggataatg gctctattgg cgaggaaaca
     1921 gactccagcc caggccggaa gaagtttccc ctcaaaatct ttaagaagcc tcttaagaag
     1981 tccggaggtg gagcatgcaa tgatgagaac tacgctcgaa ttctgcagtc gacggtaccg
     2041 cgggcccggg atccaccggt cgccaccatg gtgagcaagg gcgaggagct gttcaccggg
     2101 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     2161 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     2221 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctgggg cgtgcagtgc
     2281 ttcgcccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     2341 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     2401 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     2461 aaggaggacg gcaacatcct ggggcacaag ctggagtaca acgccatcag cgacaacgtc
     2521 tatatcaccg ccgacaagca gaagaacggc atcaaggcca acttcaagat ccgccacaac
     2581 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     2641 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccaagct gagcaaagac
     2701 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     2761 ctcggcatgg acgagctgta caagtaaagc ggccgcgact ctagatcata atcagccata
     2821 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     2881 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     2941 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     3001 gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg
     3061 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     3121 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     3181 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     3241 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     3301 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     3361 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     3421 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     3481 ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     3541 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     3601 caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc
     3661 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3721 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3781 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     3841 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     3901 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     3961 ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg
     4021 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     4081 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     4141 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa
     4201 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     4261 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     4321 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     4381 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     4441 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     4501 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc
     4561 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     4621 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     4681 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     4741 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     4801 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     4861 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     4921 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     4981 accctagggg gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta
     5041 tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg
     5101 gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat
     5161 acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc
     5221 tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt
     5281 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     5341 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     5401 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     5461 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     5521 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     5581 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     5641 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     5701 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     5761 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     5821 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     5881 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     5941 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     6001 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     6061 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc
     6121 at
//
