LOCUS       sequence-169582-        5755 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Bi-stable switch, similar to pOP512 but with a lambdaCI OR_1 
            operator in ptrc-2 promoter driving tetR..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5755)
  TITLE     Tim Lu lab plasmids
REFERENCE   2  (bases 1 to 5755)
  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: pOP549
FEATURES             Location/Qualifiers
     source          1..5755
                     /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(2070..2095,2096..2101,2102..2118,2119..2124,
                     2125..2143))
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    2100..2118
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2125..2143
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2212..2228
                     /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             2270..2893
                     /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(2343..2362)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        join(2948..2953,2954..2970,2971..2977)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2985..3001
                     /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             3052..3762
                     /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(3197..3215)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3465..3484
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      3822..3893
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(4037..4582)
                     /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(4117..4136)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(4700..4794)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4825..5619)
                     /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(4936..4955)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5546..5565
                     /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 tggcactaac gttcgtaggg gtcagtgcgt cctgctgatg tgctcagtat
     2101 ctctatcact gatagggatg tcaatctcta tcactgatag ggacggccgc ccgttccatg
     2161 gctgaaatga gctgttgaca atggagaccg ccactattat aatgtgtgga attgtgagcg
     2221 gataacaatt tcacacccgc cttcggcgaa gctagggacg agagctagca tgtctcgttt
     2281 agataaaagt aaagtgatta acagcgcatt agagctgctt aatgaggtcg gaatcgaagg
     2341 tttaacaacc cgtaaactcg cccagaagct aggtgtagag cagcctacat tgtattggca
     2401 tgtaaaaaat aagcgggctt tgctcgacgc cttagccatt gagatgttag ataggcacca
     2461 tactcacttt tgccctttag aaggggaaag ctggcaagat tttttacgta ataacgctaa
     2521 aagttttaga tgtgctttac taagtcatcg cgatggagca aaagtacatt taggtacacg
     2581 gcctacagaa aaacagtatg aaactctcga aaatcaatta gcctttttat gccaacaagg
     2641 tttttcacta gagaatgcat tatatgcact cagcgctgtg gggcatttta ctttaggttg
     2701 cgtattggaa gatcaagagc atcaagtcgc taaagaagaa agggaaacac ctactactga
     2761 tagtatgccg ccattattac gacaagctat cgaattattt gatcaccaag gtgcagagcc
     2821 agccttctta ttcggccttg aattgatcat ctgcggatta gaaaaacaac ttaaatgtga
     2881 aagtgggtct taagagctcg gactgcttaa gtcgctccat atgctcgttc ccgggcgaaa
     2941 tgagctgttg acaattaatc atccggctcg tataatgtgt ggaattgtga gcggataaca
     3001 atttcacacc aattgagtat taactatcgt tcaactgata gggaggcgcc gatggtgagc
     3061 aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa ggttcacatg
     3121 gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
     3181 gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg
     3241 gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac
     3301 atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac
     3361 ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcaaga cggcgagttc
     3421 atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag
     3481 aagactatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgcgctgaag
     3541 ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag
     3601 accacctaca aggccaagaa gcccgtgcaa ctgcccggcg cgtacaacgt caacatcaag
     3661 ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag
     3721 ggccgccact ccaccggcgg catggacgag ctgtataagt aagatatcta tcgccctagg
     3781 gaccgtctcg agagaatcaa tattaatcca acgcgtggca tcaaataaaa cgaaaggctc
     3841 agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta
     3901 ggacaatccg ccgccctaga cttagcggtc gttcgactgc ggcgagcgga aatggcttac
     3961 gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga agtgagaggg
     4021 ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac gaaatctgac
     4081 gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4141 gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc attccgctgt
     4201 tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc agttcgctcc
     4261 aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt atccggtaac
     4321 tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc agccactggt
     4381 aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac
     4441 aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt tggtagctca
     4501 gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc aagagattac
     4561 gcgcagacca aaacgatctc aagaagatca tcttattaat cagataaaat atttctagat
     4621 ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt
     4681 gttcatggct agtgcttgga ttctcaccaa taaaaaacgc ccggcggcaa ccgagcgttc
     4741 tgaacaaatc cagatggagt tctgaggtca ttactggatc tatcaacagg agtccaagcc
     4801 aattctcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat
     4861 gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc
     4921 gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac
     4981 acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg
     5041 caagcaggca tcgccgtggg tcacgacgag atcctcgccg tcgggcatac gcgccttgag
     5101 cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc
     5161 gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc
     5221 gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga
     5281 tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa
     5341 tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagccgcgc aaggaacgcc
     5401 cgtcgtggcc agccacgata gccgcgctgc ctcgtcttgc agttcattca gggcaccgga
     5461 caggtcggtc ttgacaaaaa gaaccgggcg accctgcgct gacagccgga acacggcggc
     5521 atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc
     5581 cgccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt
     5641 ctcttgatca gatcttgatc ccctgcgcca tcagatcctt ggcggcaaga aagccatcca
     5701 gtttactttg cagggcttcc caaccttacc agagggcggc ccaactggca attcc
//
