LOCUS       sequence-242343-        5800 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cytoplasmic mNeongreen mCherry tandem, ~19 % energy transfer pNM002 
            - pSAV057-mNG-mCh.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5800)
  AUTHORS   Meiresonne NY, van der Ploeg R, Hink MA, den Blaauwen T
  TITLE     Activity-Related Conformational Changes in d,d-Carboxypeptidases 
            Revealed by In Vivo Periplasmic Forster Resonance Energy Transfer 
            Assay in Escherichia coli.
  JOURNAL   MBio. 2017 Sep 12;8(5). pii: mBio.01089-17. doi: 
            10.1128/mBio.01089-17.
  PUBMED    28900026
REFERENCE   2  (bases 1 to 5800)
  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: "MBio."; 
            date: "2017-09-12"; volume: "8(5). pii: mBio.01089-17. doi"; pages: 
            " 10.1128/mBio.01089-17"
COMMENT     Sequence Label: mNG-mCh
FEATURES             Location/Qualifiers
     source          1..5800
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1186..1191,1192..1208,1209..1215)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1223..1239
                     /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)."
     primer_bind     1228..1250
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1947..2657
                     /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(2092..2110)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2360..2379
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2668..2697
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2713..2730
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2786..2803)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2956..3042
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3134..3161
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3232..3251)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3233..3251)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3258..3275)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3258..3274)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3267..3289)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        3750..3852
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             3853..4512
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3919..3938)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      4573..5118
                     /direction=RIGHT
                     /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     5019..5038
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5235..5252
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5494..5516
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        5594..5671
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             join(5672..5800,1..954)
                     /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     complement(5691..5710)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg
       61 attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt
      121 aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc
      181 gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc
      241 attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt
      301 ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat
      361 gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg
      421 ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa
      481 tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg
      541 tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg
      601 gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc
      661 gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc
      721 ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc
      781 ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg
      841 gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc
      901 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
      961 cgcaattaat gtaagttagc gcgaattgat ctggtttgac agcttatcat cgactgcacg
     1021 gtgcaccaat gcttctggcg tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc
     1081 gtaaatcact gcataattcg tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt
     1141 gcgccgacat cataacggtt ctggcaaata ttctgaaatg agctgtttac aattaatcat
     1201 ccggctcgta taatgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagacca
     1261 tggcctctct cccagcgaca catgagttac acatctttgg ctccatcaac ggtgtggact
     1321 ttgacatggt gggtcagggc accggcaatc caaatgatgg ttatgaggag ttaaacctga
     1381 agtccaccaa gggtgacctc cagttctccc cctggattct ggtccctcat atcgggtatg
     1441 gcttccatca gtacctgccc taccctgacg ggatgtcgcc tttccaggcc gcgatggtag
     1501 atggctccgg ataccaagtc catcgcacaa tgcagtttga agatggtgcc tcccttactg
     1561 ttaactaccg ctacacctac gagggaagcc acatcaaagg agaggcccag gtgaagggga
     1621 ctggtttccc tgctgacggt cctgtgatga ccaactcgct gaccgctgcg gactggtgca
     1681 ggtcgaagaa gacttacccc aacgacaaaa ccatcatcag tacctttaag tggagttaca
     1741 ccactggaaa tggcaagcgc taccggagca ctgcgcggac cacctacacc tttgccaagc
     1801 caatggcggc taactatctg aagaaccagc cgatgtacgt gttccgtaag acggagctca
     1861 agcactccaa gaccgagctc aacttcaagg agtggcaaaa ggcctttacc gatgtgatgg
     1921 gcatggacga gctgtacaag gaattcatgg tgagcaaggg cgaggaggat aacatggcta
     1981 tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt
     2041 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
     2101 aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt
     2161 acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct
     2221 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg
     2281 tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca
     2341 ccaacttccc ctccgacggc cccgtaatgc agaagaagac tatgggctgg gaggcctcct
     2401 ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc
     2461 tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg
     2521 tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg
     2581 actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg
     2641 acgagctgta caagtaagct tgggcccgaa caaaaactca tctcagaaga ggatctgaat
     2701 agcgccgtcg accatcatca tcatcatcat tgagtttaaa cggtctccag cttggctgtt
     2761 ttggcggatg agagaagatt ttcagcctga tacagattaa atcagaacgc agaagcggtc
     2821 tgataaaaca gaatttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga
     2881 actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gtctccccat gcgagagtag
     2941 ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     3001 atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg
     3061 aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg
     3121 catcaaatta agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt
     3181 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgccaattc gccctatagt
     3241 gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     3301 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     3361 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg
     3421 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3481 cttgccagcg ctgatgtccg gcggtgcttt tgccgttacg caccaccccg tcagtagctg
     3541 aacaggaggg acagctgata gaaacagaag ccactggagc acctcaaaaa caccatcata
     3601 cactaaatca gtaagttggc agcatcaccc gacgcacttt gcgccgaata aatacctgtg
     3661 acggaagatc acttcgcaga ataaataaat cctggtgtcc ctgttgatac cgggaagccc
     3721 tgggccaact tttggcgaaa atgagacgtt gatcggcacg taagaggttc caactttcac
     3781 cataatgaaa taagatcact accgggcgta ttttttgagt tatcgagatt ttcaggagct
     3841 aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat atcccaatgg
     3901 catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta taaccagacc
     3961 gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca caagttttat
     4021 ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt tcgtatggca
     4081 atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac cgttttccat
     4141 gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt ccggcagttt
     4201 ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta tttccctaaa
     4261 gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt caccagtttt
     4321 gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcactat gggcaaatat
     4381 tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca tgccgtttgt
     4441 gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga tgagtggcag
     4501 ggcggggcgt aattttttta aggcagttat tggtgcctag aaatatttta tctgattaat
     4561 aagatgatct tcttgagatc gttttggtct gcgcgtaatc tcttgctctg aaaacgaaaa
     4621 aaccgccttg cagggcggtt tttcgaaggt tctctgagct accaactctt tgaaccgagg
     4681 taactggctt ggaggagcgc agtcaccaaa acttgtcctt tcagtttagc cttaaccggc
     4741 gcatgacttc aagactaact cctctaaatc aattaccagt ggctgctgcc agtggtgctt
     4801 ttgcatgtct ttccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4861 actgaacggg gggttcgtgc atacagtcca gcttggagcg aactgcctac ccggaactga
     4921 gtgtcaggcg tggaatgaga caaacgcggc cataacagcg gaatgacacc ggtaaaccga
     4981 aaggcaggaa caggagagcg cacgagggag ccgccagggg gaaacgcctg gtatctttat
     5041 agtcctgtcg ggtttcgcca ccactgattt gagcgtcaga tttcgtgatg cttgtcaggg
     5101 gggcggagcc tatggaaaaa cggctttgcc gcggccctct cacttccctg ttaagtatct
     5161 tcctggcatc ttccaggaaa tctccgcccc gttcgtaagc catttccgct cgccgcagtc
     5221 gaacgaccga gcgtagcgag tcagtgagcg aggaagcgga atatatcctg tatcacatat
     5281 tctgctgacg caccggtgca gccttttttc tcctgccaca tgaagcactt cactgacacc
     5341 ctcatcagtg ccaacatagt aagccagtat acactccgct atcgctacgt gactgggtca
     5401 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     5461 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     5521 caccgtcatc accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag gcgaagcggc
     5581 atgcatttac gttgacacca tcgaatggtg caaaaccttt cgcggtatgg catgatagcg
     5641 cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc
     5701 agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt
     5761 ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg
//
