LOCUS       sequence-271855-        5976 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of KNL1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5976)
  TITLE     Cheeseman Lab Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5976)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pFR022
FEATURES             Location/Qualifiers
     source          1..5976
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             601..618
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(671..689)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      685..732
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      769..1224
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(856..875)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1066..1087
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1316..2131)
                     /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(1316..2131)
                     /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     2039..2058
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2123..2142
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2253..2841
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2742..2761
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2995..3012
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3027..3166
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3092..3111)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3252..3274
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3268..3459)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4268..5350)
                     /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     5312..5331
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5351..5428)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5634..5653
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5737..5756
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5737..5755
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5756..5780
                     /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             5795..5817
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5836..5853
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5863..5880
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5884..5916
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
ORIGIN
        1 gtattcacct ttgtttatga cacgatacaa ctcaccatca cctttgaaga gtcagttgtt
       61 ggtttccctt tcctggacaa gcgttatagg aagattgttg atgtcaattt tcaatctctg
      121 ttagatgagg atcaagctcc tccttcctcc cttttagttc ataagcttat tttccagtac
      181 gttgaagaaa aggaatcctg gaagaagaca tgtacaaccc agcatcagtt acccaagatg
      241 cttgaagaat tctcactggt agtgcaccat tgcagactcc ttggagagga gattgagtat
      301 ttaaagagat ggggaccaaa ttataaccta atgaacatag atattaataa taatgaattg
      361 agacttttat tctctagctc cgcagcattt gcaaagtttg aaataacttt gtttctctca
      421 gcctattatc catctgtacc attaccttcc accattcaga atcacgttgg gaacactagc
      481 caagatgata ttgctaccat tctatctaaa gtgccactgg agaacaacta cctgaagaat
      541 gtagtcaagc aaatttacca agatctgttt caggactgcc atttctacca ctagctcgag
      601 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
      661 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
      721 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
      781 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
      841 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
      901 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
      961 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     1021 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     1081 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     1141 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     1201 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1261 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga
     1321 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     1381 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     1441 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     1501 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     1561 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     1621 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     1681 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     1741 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     1801 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     1861 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     1921 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     1981 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     2041 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag
     2101 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     2161 cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     2221 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     2281 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     2341 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     2401 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     2461 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     2521 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     2581 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     2641 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     2701 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     2761 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     2821 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     2881 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     2941 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     3001 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     3061 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     3121 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     3181 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     3241 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     3301 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     3361 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     3421 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     3481 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     3541 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     3601 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     3661 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     3721 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     3781 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     3841 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     3901 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc
     3961 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     4021 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     4081 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     4141 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     4201 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     4261 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4321 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     4381 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     4441 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     4501 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     4561 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     4621 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     4681 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     4741 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     4801 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     4861 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     4921 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     4981 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     5041 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     5101 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     5161 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     5221 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     5281 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     5341 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     5401 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     5461 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     5521 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     5581 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     5641 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     5701 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat
     5761 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
     5821 accatgggca gcagccatca tcatcatcat cacagcagcg gcctggtgcc gcgcggcagc
     5881 catatggcta gcatgactgg tggacagcaa atgggtcgcg gatccatgtt gagcttgtct
     5941 gagtgggatg tcgttgagtg gagtgatgat caagct
//
