LOCUS       sequence-352676-        5969 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mSandy2 using the T7 Promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5969)
  AUTHORS   Legault S, Fraser-Halberg DP, McAnelly RL, Eason MG, Thompson MC, 
            Chica RA
  TITLE     Generation of bright monomeric red fluorescent proteins via 
            computational design of enhanced chromophore packing.
  JOURNAL   Chem Sci. 2022 Jan 11;13(5):1408-1418. doi: 10.1039/d1sc05088e. 
            eCollection 2022 Feb 2.
  PUBMED    35222925
REFERENCE   2  (bases 1 to 5969)
  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: 
            "10.1039/d1sc05088e"; journalName: "Chem Sci"; date: "2022-01-11- 
            11"; volume: "13"; issue: "5"; pages: "1408-1418"
COMMENT     Sequence Label: pET-29b(+)-mSandy2
FEATURES             Location/Qualifiers
     source          1..5969
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             4..21
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             739..756
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(809..827)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      823..870
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      907..1362
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(994..1013)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1204..1225
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1454..2269)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1454..2269)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2177..2196
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2261..2280
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2391..2979
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2880..2899
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3133..3150
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3165..3304
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3230..3249)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3390..3412
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3406..3597)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4406..5488)
                     /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     5450..5469
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5489..5566)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5772..5791
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5879..5898
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5879..5897
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5898..5922
                     /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             5937..5959
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 ggtcatcacc atcatcacca tgaggtttcc aaaggcgagg aagacaatat ggcgattatt
       61 aaggaattta tgcgtttcaa ggtacacatg gaaggaagcg tcaatggtca cgaattcgag
      121 atcgagggcg aaggagaggg tcgcccctat gagggcaccc agacggcaaa acttaaggtg
      181 accaagggcg gacccctgcc attcgcttgg gatattttgt ccctgcaatt catgtacgga
      241 tcgaaagcct atgtgaagca ccctgcggat atcccggact acttaaagct tagctttccc
      301 gagggcttta agtgggaacg tgttatgaac tttgaggatg gtggagtagt caccgttact
      361 caagacagtt cattacagga tggcgaattc atctataagg ttaagcttca tggtacgaat
      421 ttcccgtcgg acgggcctgt tatgcagaag aagactatgg gcagcgaggc gtcatcggag
      481 cgtatgtatc cagaggacgg cgcattaaag ggtgaggtta aggtgcgttt taagttaaaa
      541 gatggcggcc actacgttgc tgaagttaag acaacctaca aggcgaagaa gcctgttcaa
      601 ttgcctggag cctattacgc taacatcaag atggacatca ctagccacaa tgaggattat
      661 accatcgtgg agcagtatga gcgttgtgag ggccgtcact caacgggtgg catggacgag
      721 ctgtataagt gactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
      781 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
      841 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
      901 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
      961 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     1021 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     1081 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     1141 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     1201 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     1261 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     1321 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     1381 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     1441 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     1501 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     1561 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     1621 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     1681 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
     1741 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     1801 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
     1861 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     1921 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     1981 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     2041 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     2101 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     2161 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     2221 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
     2281 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
     2341 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     2401 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     2461 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     2521 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     2581 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     2641 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     2701 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     2761 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     2821 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     2881 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     2941 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3001 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     3061 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3121 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     3181 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     3241 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     3301 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     3361 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     3421 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     3481 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     3541 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     3601 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     3661 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     3721 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     3781 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     3841 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     3901 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     3961 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     4021 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     4081 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     4141 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     4201 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     4261 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     4321 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     4381 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4441 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     4501 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     4561 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     4621 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     4681 ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     4741 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     4801 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     4861 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     4921 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     4981 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     5041 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     5101 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     5161 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     5221 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     5281 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     5341 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     5401 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     5461 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     5521 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     5581 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     5641 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     5701 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     5761 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     5821 atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc cgcgaaatta
     5881 atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg
     5941 tttaacttta agaaggagat atacatatg
//
