LOCUS       sequence-367385-        6250 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of SNAP-ecDHFRmt fusion in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6250)
  AUTHORS   Wang L, Hiblot J, Popp C, Xue L, Johnsson K
  TITLE     Environmentally Sensitive Color-Shifting Fluorophores for 
            Bioimaging.
  JOURNAL   Angew Chem Int Ed Engl. 2020 Dec 1;59(49):21880-21884. doi: 
            10.1002/anie.202008357. Epub 2020 Sep 28.
  PUBMED    32762146
REFERENCE   2  (bases 1 to 6250)
  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"; doi: 
            "10.1002/anie.202008357"; journalName: "Angew Chem Int Ed Engl"; 
            date: "2020-12-1- 1"; volume: "59"; issue: "49"; pages: 
            "21880-21884"
COMMENT     Sequence Label: pET51b_SNAP-ecDHFRmt
FEATURES             Location/Qualifiers
     source          1..6250
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     960..979
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(999..1076)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1282..1301
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1389..1408
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1389..1407
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1408..1432
                     /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             1447..1469
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1485..1508
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1515..1529
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             2127..2603
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     CDS             2634..2663
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(2686..2704)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2700..2747
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2784..3239
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2871..2890)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3081..3102
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3354..4145,4146..4214))
                     /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     3977..3996
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4215..4307)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4388..4976
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4877..4896
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5130..5147
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5162..5301
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5227..5246)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5387..5409
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5403..5594)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6166..6250,1..998))
                     /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"
ORIGIN
        1 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
       61 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
      121 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
      181 tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
      241 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
      301 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
      361 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
      421 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
      481 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
      541 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
      601 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
      661 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
      721 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
      781 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
      841 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
      901 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
      961 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     1021 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     1081 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     1141 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     1201 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     1261 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     1321 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc
     1381 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     1441 aataattttg tttaacttta agaaggagat ataccatggc aagctggagc cacccgcagt
     1501 tcgaaaaggg tgcagatgac gacgacaagg taccgcatat ggataaagac tgcgaaatga
     1561 aacgcactac cctggacagc ccgctgggta aactggaact gagcggttgc gagcagggcc
     1621 tgcacgaaat cattttcctg ggcaaaggta cgagcgctgc ggatgcggtc gaagtaccgg
     1681 ctccggctgc ggttctgggt ggcccggagc cgctgatgca ggctaccgca tggctgaacg
     1741 catattttca ccagccagag gcgattgaag agttccctgt accggccctg catcacccgg
     1801 tatttcagca ggaatccttt acccgtcagg tactgtggaa actgctgaaa gtagtgaaat
     1861 tcggcgaggt gatctcctat tcccatctgg cggcgctggc tggcaaccca gcggcaactg
     1921 cggcggtgaa aaccgctctg tctggtaatc cggtgccaat tctgatcccg tgccaccgtg
     1981 tggttcaggg cgacctggac gtaggtggct acgaaggcgg cctggctgtg aaagaatggc
     2041 tgctggcgca cgaaggtcac cgtctgggta aaccaggcct gggcgaattc ggcggaagcg
     2101 gaggaagtgg cggctctggc ggatcgatga tcagtctgat tgcggcgtta gcggtagatc
     2161 gcgttatcgg catggaaaac gccatgccgt ggaacctgcc tgccgatctc gcctggttta
     2221 aacgcaacac cttaaataaa cccgtgatta tgggccgcca tacctgggaa tcaatcggtc
     2281 gtccgttgcc aggacgcaaa aatattatcc tcagcagtca accgggtacg gacgatcgcg
     2341 taacgtgggt gaagtcggtg gatgaagcca tcgcggcgtg tggtgacgta ccagaaatca
     2401 tggtgattgg cggcggtcgc gtttatgaac agttcttgcc aaaagcgcaa aaactgtatc
     2461 tgacgcatat cgacgcagaa gtggaaggcg acacccattt cccggattac gagccggatg
     2521 actgggaatc ggtattcagc gaattccacg atgctgatgc gcagaactct cacagctatt
     2581 gctttgagat tctggagcgg cggggagctc cgggcttctc ctcaatttcc gctcatcacc
     2641 accatcatca ccatcaccac cactaattaa cctaggctgc tgccaccgct gagcaataac
     2701 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     2761 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2821 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2881 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2941 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3001 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     3061 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     3121 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     3181 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc
     3241 tggcggcacg atggcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     3301 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     3361 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     3421 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     3481 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     3541 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     3601 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     3661 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     3721 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     3781 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     3841 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     3901 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     3961 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     4021 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     4081 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     4141 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     4201 tgttgaatac tcatactctt cctttttcaa tcatgattga agcatttatc agggttattg
     4261 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gtcatgacca
     4321 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     4381 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4441 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4501 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     4561 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4621 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4681 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4741 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     4801 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     4861 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     4921 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     4981 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     5041 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     5101 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     5161 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     5221 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     5281 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     5341 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     5401 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     5461 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     5521 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     5581 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     5641 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     5701 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     5761 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     5821 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     5881 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     5941 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     6001 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gctagtcatg
     6061 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     6121 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     6181 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     6241 ggtttgcgta
//
