LOCUS       sequence-436378-        4323 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Caging at lumenal N-ter of POI: TMEM-PhoCl-MCS in pTREtight2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4323)
  AUTHORS   Kashyap P, Bertelli S, Cao F, Kostritskaia Y, Blank F, Srikanth NA, 
            Schlack-Leigers C, Saleppico R, Bierhuizen D, Lu X, Nickel W, 
            Campbell RE, Plested AJR, Stauber T, Taylor MJ, Ewers H
  TITLE     An optogenetic method for the controlled release of single 
            molecules.
  JOURNAL   Nat Methods. 2024 Mar 8. doi: 10.1038/s41592-024-02204-x.
  PUBMED    38459384
REFERENCE   2  (bases 1 to 4323)
  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: "Nat 
            Methods. 2024 Mar 8. doi: 10.1038/s41592-024-02204-x."
COMMENT     Sequence Label: pRER-PhoCl-Insert
FEATURES             Location/Qualifiers
     source          1..4323
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(63..651)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(143..162)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(822..1613,1614..1682))
                     /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     1445..1464
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1683..1787)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1855..1873
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1911..1933)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     polyA_signal    2036..2117
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     2061..2080
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(3709..4023)
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     primer_bind     complement(3712..3736)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    3783..3801
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3819..3837
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3854..3872
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3890..3908
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3926..3944
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3961..3979
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3997..4015
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(join(4032..4038,4039..4056,4057..4062))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4077..4098
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4215..4232)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
       61 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
      121 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
      181 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
      241 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
      301 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
      361 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
      421 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
      481 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
      541 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
      601 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
      661 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
      721 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
      781 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
      841 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
      901 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
      961 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     1021 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     1081 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     1141 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     1201 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1261 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1321 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1381 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1441 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1501 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1561 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1621 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1681 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     1741 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     1801 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     1861 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     1921 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     1981 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaactc tcgaggcagt
     2041 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
     2101 gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg
     2161 aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatg gctgattatg
     2221 atcctctaga gatatcgtcg acaagcttat cgatgcggcc gcgctagctg ccgccgccgc
     2281 ctccgccact acctcctgtg ccgcctccgc ctctagggta ctttgtgaac acccggttgc
     2341 cgtagtggaa ggcggtggtc agaatgtcgt aggcgaaagg cagaggggcg ccttctttca
     2401 cttccaggtc gatggtctgg atgccctcaa agggcttgcc gctgccttcg ccttcgatca
     2461 caaaggcgtg gccgttcacg ttgccttcca tccgcagctt gttcttcatg tcgggcttga
     2521 tcacgctggt gattgtctct tcgcccttag acaccatgcc gccgctgccg cctttgtaca
     2581 gctcgtccat agaatcggtg gaggtcttgg ccacggcgtg ctcgtacagc ttgactttgt
     2641 tgtagtcctt gtcgtggctc aggatctcga tccggtgatc cacgaagtgg cagtcgggca
     2701 gcttcacggg tttctgcttc actttgtagg tggtccggta gtcgcctctg tagtgtccgc
     2761 cgcctttcag cagcagcttc attttcacgt cgcccttcag cacgccatct cgctcgtaca
     2821 tcttctcggt gctggcttcc cagccaactg ttctcttctg catcacgggg ccgtttggag
     2881 ggaagttcgt gccctgaaag tggatcttgt tgatgaagct gtcgccttcc atggtgatgt
     2941 cgttggtggc gatacagatg ccgccgtcct cgtatgtcat ggatctctcc cagctgtagc
     3001 cctcggggaa gctctgcttg aagtagtcgg gtatgacctc gagatctgag tccggacttg
     3061 tacagctggc gaaggctttt ccggcatcct ctttgcctct gtaccgcctc ttgccgtgtg
     3121 tgaaggggat gtagcggtac ttgatcatgt gcttgatctg gcgcttcatg gtgatacaga
     3181 acagcatgat gaagtacatg accaggatgg gccagaacac gggcacgttg aaggcgtcga
     3241 agaaggtgca gaccatagca accaggatgc ccttggtggc ggcgtgccaa aatttgaact
     3301 cgggcagccg ccggatgaag ggtctgaatt cctcgttctg cttggtaggc aggctaggtc
     3361 catcgtcgga atcttccatc aggctggggt ccaccttggg agacagaaag gcaatgaaca
     3421 ggttcaggtg gtagattccc agggcgtatg tcacgatgta ccagccttgc agcaggtaca
     3481 cccggatcat gtacacgaag ctcaggccca gtgtcacaac ccatctgacg gcggtgtaag
     3541 gggtgctctt atccagccag ctctggtaga tctggcccag ccgggtaaag aaccggtaca
     3601 ccacgctagg cttgccgtgc acagactctc ccacagaatc gccttcagac atggtggcca
     3661 cgcgtcagct gactagagga tccccgggta ccgagctcga attctccagg cgatctgacg
     3721 gttcactaaa cgagctctgc ttatataggc ctcccaccgt acacgcctac ctcgacatac
     3781 gttctctatc actgataggg agtaaactcg acatacgttc tctatcactg atagggataa
     3841 actcgacata cgttctctat cactgatagg gagtaaactc gacatacgtt ctctatcact
     3901 gatagggagt aaactcgaca tacgttctct atcactgata gggagtaaac tcgacatcgt
     3961 tctctatcac tgatagggag taaactcgac atacgttctc tatcactgat agggagtaaa
     4021 ctcgagccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     4081 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4141 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4201 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4261 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4321 aag
//
