LOCUS       sequence-135321-        5866 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Reporter plasmid encoding mCherry, codon optimized for E. coli, 
            transcriptionally driven by orthogonal T7-lac promoter variant 4F2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5866)
  AUTHORS   Cress BF, Jones JA, Kim DC, Leitz QD, Englaender JA, Collins SM, 
            Linhardt RJ, Koffas MA
  TITLE     Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible
            hybrid T7-lac promoters for modular, tuneable control of metabolic 
            pathway fluxes in Escherichia coli.
  JOURNAL   Nucleic Acids Res. 2016 Apr 13. pii: gkw231.
  PUBMED    27079979
REFERENCE   2  (bases 1 to 5866)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2016 Apr 13. pii: gkw231."
COMMENT     Sequence Label: pETM6-4F2-mCherry
FEATURES             Location/Qualifiers
     source          1..5866
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     RBS             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             801..845
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(884..902)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      898..945
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      989..1444
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     1286..1307
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1559..2350,2351..2419))
                     /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     2182..2201
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2420..2512)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2593..3181
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3082..3101
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3335..3352
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3367..3509
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3435..3454)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3595..3617
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3611..3802)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4374..5456)
                     /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     5418..5437
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5457..5534)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5740..5759
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatataca tatggtttca aaaggcgaag aagacaacat ggcgattatc aaggaattta
      121 tgcgtttcaa ggtccacatg gaaggcagcg tcaatggtca cgaatttgaa attgaaggcg
      181 aaggtgaagg ccgtccgtat gaaggcaccc agacggcaaa actgaaggtc accaaaggcg
      241 gtccgctgcc gtttgcttgg gatattctgt caccgcaatt catgtatggt tcgaaagcgt
      301 acgttaagca tccggccgat atcccggact atctgaaact gtcctttccg gaaggcttca
      361 aatgggaacg tgttatgaac ttcgaagatg gcggtgtggt taccgtcacg caggatagct
      421 ctctgcaaga cggtgaattt atttataaag tgaagctgcg cggcaccaat ttcccgagcg
      481 atggtccggt tatgcagaaa aagacgatgg gctgggaagc gagttccgaa cgtatgtacc
      541 cggaagacgg tgccctgaaa ggcgaaatca agcagcgcct gaaactgaag gatggcggtc
      601 actatgacgc agaagtgaaa accacgtaca aggctaaaaa gccggtccaa ctgccgggtg
      661 catacaacgt gaacatcaag ctggatatca ccagccataa cgaagactat acgatcgttg
      721 aacagtacga acgtgcagaa ggccgccact ctaccggcgg tatggatgaa ctgtacaaat
      781 aaggtaccct cgagtctggt aaagaaaccg ctgctgcgaa atttgaacgc cagcacatgg
      841 actcgtctac tagtcgcagc ttaattaacc taaactgctg ccaccgctga gcaataacta
      901 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctagc gaaaggagga
      961 gtcgactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     1021 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     1081 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     1141 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     1201 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     1261 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     1321 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     1381 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     1441 atttctggcg gcacgatggc atgagattat caaaaaggat cttcacctag atccttttaa
     1501 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     1561 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     1621 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     1681 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     1741 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     1801 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     1861 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     1921 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     1981 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     2041 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     2101 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     2161 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     2221 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     2281 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     2341 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     2401 ggaaatgttg aatactcata ctcttccttt ttcaatcatg attgaagcat ttatcagggt
     2461 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggtcat
     2521 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     2581 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     2641 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     2701 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     2761 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     2821 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     2881 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     2941 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3001 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3061 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3121 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3181 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3241 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3301 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3361 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatata
     3421 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     3481 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     3541 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     3601 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     3661 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     3721 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     3781 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     3841 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     3901 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     3961 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     4021 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     4081 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     4141 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     4201 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     4261 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     4321 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     4381 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4441 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     4501 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     4561 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     4621 ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac
     4681 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     4741 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     4801 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     4861 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     4921 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     4981 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     5041 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     5101 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     5161 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     5221 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     5281 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     5341 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     5401 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     5461 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     5521 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     5581 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     5641 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     5701 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     5761 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagcctag
     5821 gatcgagatc gatctcgatc ccgcgaaatt aatacgactc actact
//
