LOCUS       sequence-233934-        5807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mKelly2 tagged N-terminally with an MHHHHHHGSG tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5807)
  AUTHORS   Wannier TM, Gillespie SK, Hutchins N, McIsaac RS, Wu SY, Shen Y, 
            Campbell RE, Brown KS, Mayo SL
  TITLE     Monomerization of far-red fluorescent proteins.
  JOURNAL   Proc Natl Acad Sci U S A. 2018 Nov 27;115(48):E11294-E11301. doi: 
            10.1073/pnas.1807449115. Epub 2018 Nov 13.
  PUBMED    30425172
REFERENCE   2  (bases 1 to 5807)
  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: 
            "10.1073/pnas.1807449115"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2018-11-27- 27"; volume: "115"; issue: "48"; pages: 
            "E11294-E11301"
COMMENT     Sequence Label: pET-mKelly2
FEATURES             Location/Qualifiers
     source          1..5807
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     921..940
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(960..1037)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1243..1262
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1350..1369
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1350..1368
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1369..1393
                     /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             1408..1430
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1440..1457
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2154..2177
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(2204..2222)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2218..2265
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2302..2757
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2389..2408)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2599..2620
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2872..3663,3664..3732))
                     /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     3495..3514
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3733..3825)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3906..4494
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4395..4414
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4648..4665
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4680..4819
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4745..4764)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4905..4927
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4921..5112)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5684..5807,1..959))
                     /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 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
       61 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
      121 gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg
      181 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
      241 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
      301 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
      361 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
      421 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
      481 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
      541 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
      601 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
      661 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
      721 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
      781 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
      841 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
      901 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
      961 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     1021 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     1081 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     1141 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     1201 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     1261 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     1321 gatcgagatc gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
     1381 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgc
     1441 accatcatca ccaccacggt tctggtgaac tgattaaaga aaacatgcgt atgaaactgt
     1501 acatggaagg taccgttggc aaccatcact tcaaatgcac cgcggaaggt gagggtaaac
     1561 cgtacgaagg cacccagacc cagcgtatta aagttgttga aggtggtccg ctgccgttcg
     1621 cgttcgacat cctggcgacc tgcttcatgt atggttctaa aacttttatc aactaccctc
     1681 agggtatccc ggacttcttc aaacagtctt tcccagaagg tttcacctgg gaacgtgtta
     1741 ccacctacga agacggcggt gttctgaccg ttacccagga cacctctctg caggacggtt
     1801 acctgatcta caacgttaaa ctgcgtggtg ttaacttccc gtctaacggc cctgttatgc
     1861 agaaaaagac tctgggttgg gaaccgacca ccgagtacct ggtcccggtt gacggtggcc
     1921 tggagggtac atgtgatatg gcgctgcgtc tggttggtgg cggtcacctg aagtgcaatc
     1981 tgaaaaccac gtatcgttcc aaaaaaccag cgaaaaacct gaagatgccg ggtgagcacc
     2041 aggttgaccg taagctggag cgtatcaaag aggcggacaa cgaaacctac gttgaacagc
     2101 acgaagttgc ggttgcgaga taatgagctt tcttgtacaa agtggtttcc gcttggagcc
     2161 acccgcagtt cgaaaagtaa gtgattaacc taggctgctg ccaccgctga gcaataacta
     2221 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     2281 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2341 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     2401 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2461 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     2521 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     2581 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     2641 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     2701 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttctg
     2761 gcggcacgat ggcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     2821 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     2881 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     2941 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     3001 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     3061 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     3121 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     3181 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     3241 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     3301 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     3361 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     3421 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     3481 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     3541 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     3601 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     3661 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     3721 ttgaatactc atactcttcc tttttcaatc atgattgaag catttatcag ggttattgtc
     3781 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggt catgaccaaa
     3841 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3901 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3961 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4021 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     4081 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     4141 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4201 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4261 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     4321 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     4381 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     4441 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     4501 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     4561 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     4621 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     4681 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     4741 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     4801 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     4861 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     4921 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     4981 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     5041 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     5101 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     5161 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     5221 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     5281 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     5341 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     5401 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     5461 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     5521 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc tagtcatgcc
     5581 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     5641 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     5701 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5761 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacg
//
