LOCUS       sequence-467867-        6248 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden gate domesticated pET28b for scarless cloning with His6-3C.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6248)
  AUTHORS   Gaur D, Wohlever ML
  TITLE     A suite of pre-assembled, pET28b-based Golden Gate vectors for 
            efficient protein engineering and expression.
  JOURNAL   Protein Sci. 2025 Apr;34(4):e70106. doi: 10.1002/pro.70106.
  PUBMED    40130802
REFERENCE   2  (bases 1 to 6248)
  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: "Protein 
            Sci."; date: "2025-04"; volume: "34(4):e70106. doi"; pages: " 
            10.1002/pro.70106"
COMMENT     Sequence Label: p277_DG
FEATURES             Location/Qualifiers
     source          1..6248
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     461..480
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(500..577)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     783..802
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     886..905
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        886..904
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    905..929
                     /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             944..966
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             985..1002
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1012..1035
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(1094..1780)
                     /codon_start=1
                     /product="bright cysteine-free monomeric red fluorescent 
                     protein with fast and complete maturation (Gadella et al., 
                     2023)"
                     /label=mScarlet3
                     /translation="MDSTEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LRVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYWKQSFPEGFKWERVMNFEDGGA
                     VSVAQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDVVLKGDIKM
                     ALRLKDGGRYLADFKTTYRAKKPVQMPGAFNIDRKLDITSHNEDYTVVEQYERSVARHS
                     TGGSGGS"
     RBS             1787..1798
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    1859..1880
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             1998..2015
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2068..2086)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2082..2129
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2166..2621
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2253..2272)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2463..2484
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2713..3528)
                     /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(2713..3528)
                     /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     3436..3455
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3520..3539
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3650..4238
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4139..4158
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4392..4409
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4424..4563
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4489..4508)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4649..4671
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4665..4856)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5665..6248,1..499))
                     /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 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
       61 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
      121 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
      181 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
      241 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
      301 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
      361 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
      421 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
      481 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
      541 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
      601 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
      661 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
      721 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
      781 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
      841 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta
      901 taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga
      961 aggagatata ccatgggcag cagccatcat catcatcatc acagcagcgg cctggaagtg
     1021 ctcttccagg gcccgggatc cagagaccgc ctggctctag tagcgatcta cactagcact
     1081 atcagcgtta ttagctgccg ccgctgccgc cggtgctatg gcgcgccacg ctgcgttcat
     1141 actgttccac cacggtataa tcttcgttat ggctcgtaat atccagtttg cgatcaatgt
     1201 taaacgcgcc cggcatctgc accggttttt tcgcgcgata ggtggtttta aaatccgcca
     1261 gatagcggcc gccatctttc aggcgcagcg ccattttaat atcgcctttc agcaccacat
     1321 cttccggata caggcgttcg gtgctcgctt cccagcccat cgtttttttc tgcatcaccg
     1381 ggccatccgg cggaaagttg gtgccgcgca gtttcacttt ataaatcagg gtgccatctt
     1441 ccaggctcgt atcttgcgcc acgctcaccg cgccgccatc ttcaaagttc atcacgcgtt
     1501 cccatttaaa gccttccgga aagctctgtt tccaataatc cggaatatcc gccggatgtt
     1561 tggtaaacgc gcggctgcca tacataaact gcgggctcag aatatcccag ctaaacggca
     1621 gcgggccgcc tttggtcacg cgcagtttcg cggtctgcgt gccttcatac gggcggcctt
     1681 cgccttcgcc ttcaatttca aattcatggc cgttcatgct gccttccata tgcactttaa
     1741 agcgcataaa ttctttaatc accgcttcgg tgctatccat ctagtatttc tcctctttct
     1801 ctagtatgtg tacacaacat acggccggaa gcataaagtg taaagcctgg ggtgcctaat
     1861 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     1921 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     1981 ggtctcatga gctcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
     2041 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     2101 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     2161 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2221 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     2281 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     2341 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     2401 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     2461 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     2521 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     2581 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     2641 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     2701 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     2761 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     2821 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     2881 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     2941 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     3001 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     3061 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     3121 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     3181 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     3241 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     3301 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     3361 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     3421 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     3481 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     3541 attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt
     3601 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     3661 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     3721 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     3781 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     3841 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     3901 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     3961 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     4021 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     4081 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     4141 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     4201 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     4261 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     4321 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     4381 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     4441 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     4501 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     4561 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     4621 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     4681 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     4741 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     4801 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     4861 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     4921 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     4981 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     5041 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     5101 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     5161 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     5221 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     5281 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     5341 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     5401 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     5461 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     5521 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     5581 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     5641 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5701 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     5761 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     5821 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     5881 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     5941 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     6001 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     6061 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     6121 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     6181 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     6241 cagtcgcg
//
