LOCUS       sequence-169580-        5748 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Bi-stable switch. From Collins lab but the origin substituted for a 
            P15A origin..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5748)
  TITLE     Tim Lu lab plasmids
REFERENCE   2  (bases 1 to 5748)
  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"
COMMENT     Sequence Label: pOP512
FEATURES             Location/Qualifiers
     source          1..5748
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(58..771)
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(98..118)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     713..741
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     promoter        complement(join(823..848,849..854,855..871,872..877,
                     878..896))
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    853..871
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    878..896
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             complement(939..2021)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     1983..2002
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(2058..2083,2084..2089,2090..2106,2107..2112,
                     2113..2131))
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    2088..2106
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2113..2131
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        join(2163..2168,2169..2185,2186..2192)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2200..2216
                     /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)."
     CDS             2263..2886
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(2336..2355)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        join(2941..2946,2947..2963,2964..2970)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2978..2994
                     /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)."
     CDS             3045..3755
                     /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(3190..3208)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3458..3477
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      3815..3886
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(4030..4575)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(4110..4129)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(4693..4787)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4818..5612)
                     /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(4929..4948)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5539..5558
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 gacgtctgtg caagtactac tgttctgcag tcacttgaat tcgataccca gctgttattt
       61 gtatagttca tccatgccat gtgtaatccc agcagcggtt acaaactcaa gaaggaccat
      121 gtggtctctc ttttcgttgg gatctttcga aagggcagat tgtgtggaca ggtaatggtt
      181 gtctggtaaa aggacagggc catcgccaat cggagtattt tgttgataat ggtctgctag
      241 ttgaacgctt ccatcttcaa tgttgtgtct aattttgaag ttaactttga ttccattctt
      301 ttgtttgtct gccatgatgt atacattgtg tgagttatag ttgtattcca atttgtgtcc
      361 aagaatgttt ccatcttctt taaaatcaat accttttaac tcgattctat taacaagggt
      421 atcaccttca aacttgactt cagcacgtgt cttgtagttc ccgtcatctt tgaaaaatat
      481 agttctttcc tgtacgtaac cttcgggcat ggcactcttg aaaaagtcat gctgtttcat
      541 gtgatctggg tatctcgcaa agcattgaac accataaccg aaagtagtga caagtgttgg
      601 ccacggaaca ggtagttttc cagtagtgca aataaattta agggtaagtt ttccgtatgt
      661 tgcatcacct tcaccctctc cactgacaga aaatttgtgc ccattaacat caccatctaa
      721 ttcaacaaga attgggacaa ctccagtgaa aagttcttct cctttacgca tgtgcactac
      781 ctccttggtt gtctatgcta tgctgatcta caactggcat gcggtcagtg cgtcctgctg
      841 atgtgctcag tatctctatc actgataggg atgtcaatct ctatcactga tagggaaacg
      901 tttcgcagaa gcttccgcaa ggtaccactt tgccgcggtt actgcccgct ttccagtcgg
      961 gaaacctgtc gtgccagttg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     1021 gtattgggag ccagggtggt ttttcttttc accagtgaga cgggcaacag ttgattgccc
     1081 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     1141 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     1201 tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc
     1261 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     1321 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     1381 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     1441 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     1501 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     1561 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     1621 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ctgcgcgaga
     1681 agattgtgaa ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     1741 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     1801 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     1861 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     1921 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     1981 ccggcatact ctgcgacatc gtacagcgtt actggtttca ttgtacaccc tctccttata
     2041 ttgggaccta tggatccggt cagtgcgtcc tgctgatgtg ctcagtatct ctatcactga
     2101 tagggatgtc aatctctatc actgataggg acggccgccc gttccatggc tgaaatgagc
     2161 tgttgacaat taatcatccg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc
     2221 acacgtcgac cgccttcggc gaagctaggg acgagagcta gcatgtctcg tttagataaa
     2281 agtaaagtga ttaacagcgc attagagctg cttaatgagg tcggaatcga aggtttaaca
     2341 acccgtaaac tcgcccagaa gctaggtgta gagcagccta cattgtattg gcatgtaaaa
     2401 aataagcggg ctttgctcga cgccttagcc attgagatgt tagataggca ccatactcac
     2461 ttttgccctt tagaagggga aagctggcaa gattttttac gtaataacgc taaaagtttt
     2521 agatgtgctt tactaagtca tcgcgatgga gcaaaagtac atttaggtac acggcctaca
     2581 gaaaaacagt atgaaactct cgaaaatcaa ttagcctttt tatgccaaca aggtttttca
     2641 ctagagaatg cattatatgc actcagcgct gtggggcatt ttactttagg ttgcgtattg
     2701 gaagatcaag agcatcaagt cgctaaagaa gaaagggaaa cacctactac tgatagtatg
     2761 ccgccattat tacgacaagc tatcgaatta tttgatcacc aaggtgcaga gccagccttc
     2821 ttattcggcc ttgaattgat catctgcgga ttagaaaaac aacttaaatg tgaaagtggg
     2881 tcttaagagc tcggactgct taagtcgctc catatgctcg ttcccgggcg aaatgagctg
     2941 ttgacaatta atcatccggc tcgtataatg tgtggaattg tgagcggata acaatttcac
     3001 accaattgag tattaactat cgttcaactg atagggaggc gccgatggtg agcaagggcg
     3061 aggaggataa catggccatc atcaaggagt tcatgcgctt caaggttcac atggagggct
     3121 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
     3181 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc
     3241 tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg
     3301 actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
     3361 acggcggcgt ggtgaccgtg acccaggact cctccctgca agacggcgag ttcatctaca
     3421 aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacta
     3481 tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgcgctg aagggcgaga
     3541 tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct
     3601 acaaggccaa gaagcccgtg caactgcccg gcgcgtacaa cgtcaacatc aagttggaca
     3661 tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc
     3721 actccaccgg cggcatggac gagctgtata agtaagatat ctatcgccct agggaccgtc
     3781 tcgagagaat caatattaat ccaacgcgtg gcatcaaata aaacgaaagg ctcagtcgaa
     3841 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaat
     3901 ccgccgccct agacttagcg gtcgttcgac tgcggcgagc ggaaatggct tacgaacggg
     3961 gcggagattt cctggaagat gccaggaaga tacttaacag ggaagtgaga gggccgcggc
     4021 aaagccgttt ttccataggc tccgcccccc tgacaagcat cacgaaatct gacgctcaaa
     4081 tcagtggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggcggctc
     4141 cctcgtgcgc tctcctgttc ctgcctttcg gtttaccggt gtcattccgc tgttatggcc
     4201 gcgtttgtct cattccacgc ctgacactca gttccgggta ggcagttcgc tccaagctgg
     4261 actgtatgca cgaacccccc gttcagtccg accgctgcgc cttatccggt aactatcgtc
     4321 ttgagtccaa cccggaaaga catgcaaaag caccactggc agcagccact ggtaattgat
     4381 ttagaggagt tagtcttgaa gtcatgcgcc ggttaaggct aaactgaaag gacaagtttt
     4441 ggtgactgcg ctcctccaag ccagttacct cggttcaaag agttggtagc tcagagaacc
     4501 ttcgaaaaac cgccctgcaa ggcggttttt tcgttttcag agcaagagat tacgcgcaga
     4561 ccaaaacgat ctcaagaaga tcatcttatt aatcagataa aatatttcta gatttcagtg
     4621 caatttatct cttcaaatgt agcacctgaa gtcagcccca tacgatataa gttgttcatg
     4681 gctagtgctt ggattctcac caataaaaaa cgcccggcgg caaccgagcg ttctgaacaa
     4741 atccagatgg agttctgagg tcattactgg atctatcaac aggagtccaa gccaattctc
     4801 gaaccccaga gtcccgctca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc
     4861 gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc
     4921 tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc
     4981 cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag
     5041 gcatcgccgt gggtcacgac gagatcctcg ccgtcgggca tacgcgcctt gagcctggcg
     5101 aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga
     5161 ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg
     5221 caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc
     5281 tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc
     5341 cagtcccttc ccgcttcagt gacaacgtcg agcacagccg cgcaaggaac gcccgtcgtg
     5401 gccagccacg atagccgcgc tgcctcgtct tgcagttcat tcagggcacc ggacaggtcg
     5461 gtcttgacaa aaagaaccgg gcgaccctgc gctgacagcc ggaacacggc ggcatcagag
     5521 cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agccgccgga
     5581 gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga
     5641 tcagatcttg atcccctgcg ccatcagatc cttggcggca agaaagccat ccagtttact
     5701 ttgcagggct tcccaacctt accagagggc ggcccaactg gcaattcc
//
