LOCUS       sequence-367386-        6250 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of SNAP-ecDHFR 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-ecDHFR
FEATURES             Location/Qualifiers
     source          1..6250
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..21
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2..20
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    21..45
                     /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             60..82
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             98..121
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             128..142
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             740..1216
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGLQ
                     TWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     CDS             1247..1276
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(1299..1317)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1313..1360
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1397..1852
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1484..1503)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1694..1715
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1967..2758,2759..2827))
                     /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     2590..2609
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2828..2920)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3001..3589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3490..3509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3743..3760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3775..3914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3840..3859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4000..4022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4016..4207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4779..5861)
                     /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     5823..5842
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5862..5939)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6145..6164
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt
       61 ttgtttaact ttaagaagga gatataccat ggcaagctgg agccacccgc agttcgaaaa
      121 gggtgcagat gacgacgaca aggtaccgca tatggataaa gactgcgaaa tgaaacgcac
      181 taccctggac agcccgctgg gtaaactgga actgagcggt tgcgagcagg gcctgcacga
      241 aatcattttc ctgggcaaag gtacgagcgc tgcggatgcg gtcgaagtac cggctccggc
      301 tgcggttctg ggtggcccgg agccgctgat gcaggctacc gcatggctga acgcatattt
      361 tcaccagcca gaggcgattg aagagttccc tgtaccggcc ctgcatcacc cggtatttca
      421 gcaggaatcc tttacccgtc aggtactgtg gaaactgctg aaagtagtga aattcggcga
      481 ggtgatctcc tattcccatc tggcggcgct ggctggcaac ccagcggcaa ctgcggcggt
      541 gaaaaccgct ctgtctggta atccggtgcc aattctgatc ccgtgccacc gtgtggttca
      601 gggcgacctg gacgtaggtg gctacgaagg cggcctggct gtgaaagaat ggctgctggc
      661 gcacgaaggt caccgtctgg gtaaaccagg cctgggcgaa ttcggcggaa gcggaggaag
      721 tggcggctct ggcggatcga tgatcagtct gattgcggcg ttagcggtag atcgcgttat
      781 cggcatggaa aacgccatgc cgtggaacct gcctgccgat ctcgcctggt ttaaacgcaa
      841 caccttaaat aaacccgtga ttatgggcct ccaaacctgg gaatcaatcg gtcgtccgtt
      901 gccaggacgc aaaaatatta tcctcagcag tcaaccgggt acggacgatc gcgtaacgtg
      961 ggtgaagtcg gtggatgaag ccatcgcggc gtgtggtgac gtaccagaaa tcatggtgat
     1021 tggcggcggt cgcgtttatg aacagttctt gccaaaagcg caaaaactgt atctgacgca
     1081 tatcgacgca gaagtggaag gcgacaccca tttcccggat tacgagccgg atgactggga
     1141 atcggtattc agcgaattcc acgatgctga tgcgcagaac tctcacagct attgctttga
     1201 gattctggag cggcggggag ctccgggctt ctcctcaatt tccgctcatc accaccatca
     1261 tcaccatcac caccactaat taacctaggc tgctgccacc gctgagcaat aactagcata
     1321 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     1381 cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     1441 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     1501 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     1561 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     1621 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     1681 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     1741 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     1801 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat ttctggcggc
     1861 acgatggcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     1921 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     1981 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     2041 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     2101 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     2161 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     2221 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     2281 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     2341 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     2401 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     2461 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     2521 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     2581 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     2641 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     2701 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     2761 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     2821 tactcatact cttccttttt caatcatgat tgaagcattt atcagggtta ttgtctcatg
     2881 agcggataca tatttgaatg tatttagaaa aataaacaaa taggtcatga ccaaaatccc
     2941 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3001 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3061 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     3181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     3241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     3721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     3781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     3841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     3901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     3961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     4021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     4081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     4141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     4201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     4261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     4321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     4381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     4441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     4501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     4561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     4621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgctagtc atgccccgcg
     4681 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     4741 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4801 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4861 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     4921 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     4981 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     5041 tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc
     5101 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     5161 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     5221 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     5281 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     5341 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     5401 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     5461 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     5521 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     5581 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     5641 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     5701 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     5761 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     5821 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     5881 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     5941 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     6001 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     6061 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     6121 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     6181 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tcgatctcga
     6241 tcccgcgaaa
//
