LOCUS       pSUMO-Metap1            6733 bp DNA     circular SYN 24-JAN-2023
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6733)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 24, 2023 from SnapGene 6.1.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6733
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          432..1589
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    40..64
                     /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             79..101
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             114..131
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             138..425
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated and modified)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /note="modified to prevent cleavage by eukaryotic SUMO 
                     proteases"
                     /translation="DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRR
                     LMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG"
     primer_bind     366..390
                     /label=SeqSUMO_for
     primer_bind     403..425
                     /label=KE104a
     CDS             432..1589
                     /codon_start=1
                     /label=METAP1
                     /translation="MAAVETRVCETDGCSSEAKLQCPTCIKLGIQGSYFCSQECFKGSW
                     ATHKLLHKKAKDEKAKREVCSWTVEGDVNTDPWAGYRYTGKLRPHYPLMPTRPVPSYIQ
                     RPDYADHPLGMSESEQALKGTSQIKLLSSEDIEGMRLVCRLAREVLDIAAGMIKAGVTT
                     EEIDHAVHLACIARNCYPSPLNYYNFPKSCCTSVNEVICHGIPDRRPLQEGDIVNVDIT
                     LYRNGYHGDLNETFFVGDVDEGARKLVQTTYECLMQAIDAVKPGVRYRELGNIIQKHAQ
                     ANGFSVVRSYCGHGIHKLFHTAPNVPHYAKNKAVGVMKSGHVFTIEPMICEGGWQDETW
                     PDGWTAVTRDGKRSAQFEHTLLVTDTGCEILTRRLDSSRPHFMSQF"
     primer_bind     609..623
                     /label=KE104a
     misc_feature    1468
                     /label=mutation
     primer_bind     complement(1570..1592)
                     /label=KE112
     primer_bind     complement(1570..1592)
                     /label=KE112a
     primer_bind     complement(1570..1592)
                     /label=KE121
     CDS             1614..1631
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1642..1665)
                     /label=SeqSUMO_rev
     terminator      1698..1745
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1782..2237
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(2330..3145)
                     /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"
     rep_origin      3267..3855
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    4041..4183
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4285..4476)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5285..6367)
                     /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"
     promoter        complement(6368..6445)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
       61 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggtcatcacc
      121 atcatcatca cgggtcggac tcagaagtca atcaagaagc taagccagag gtcaagccag
      181 aagtcaagcc tgagactcac atcaatttaa aggtgtccga tggatcttca gagatcttct
      241 tcaagatcaa aaagaccact cctttaagaa ggctgatgga agcgttcgct aaaagacagg
      301 gtaaggaaat ggactcctta agattcttgt acgacggtat tagaattcaa gctgatcagg
      361 cccctgaaga tttggacatg gaggataacg atattattga ggctcaccgc gaacagattg
      421 gaggatccca tatggcggcg gtggagacgc gggtgtgcga gacggacggc tgcagcagcg
      481 aggccaagct ccagtgtccc acctgcatca agctgggcat ccagggctcg tacttctgct
      541 cgcaggaatg ctttaaagga agctgggcca ctcacaagtt actacataag aaagcaaaag
      601 atgaaaaggc aaagcgagaa gtgtgttcct ggactgtaga aggtgatgtc aacactgacc
      661 catgggcagg ctatcggtac actggtaaac tcaggccaca ttatccactg atgccaacac
      721 ggccagtgcc cagttacatt cagaggccgg actatgctga ccatccctta ggaatgtctg
      781 aatcggagca ggctcttaaa ggtacctctc agattaagtt actctcatct gaagatatag
      841 aagggatgcg actcgtgtgt aggcttgcca gagaagtttt ggacattgct gctgggatga
      901 ttaaagcggg tgtaactact gaagagatcg accacgctgt acacttagca tgcattgcaa
      961 gaaattgcta tccttctccc ctgaattact ataacttccc aaagtcttgt tgtacctcag
     1021 tgaatgaagt catctgccat gggattccag acaggcggcc tttgcaagaa ggtgatattg
     1081 ttaatgtgga catcactctt tatcgaaatg gttaccatgg ggatctaaat gagacctttt
     1141 tcgttggcga tgtggatgaa ggagcacgga aacttgttca gactacatat gagtgcctga
     1201 tgcaagccat cgatgcagtg aagcctggtg ttcgatacag agaactggga aacatcattc
     1261 agaaacacgc ccaagcaaat ggattctcag ttgttcgaag ctattgtggg catgggatcc
     1321 acaaactttt ccatacagcc cccaatgtcc cgcactatgc caaaaataaa gcagttggtg
     1381 tgatgaagtc gggtcatgtg tttacaattg agccaatgat ttgtgaaggt ggatggcagg
     1441 atgagacttg gccagatggc tggactgcag tgacgaggga cgggaagcga tctgctcagt
     1501 ttgagcacac cctgctggtc acggacactg gctgtgaaat tctcacccgg agactcgata
     1561 gctctcggcc tcatttcatg tcccagtttt aaaagcttgc ggccgcactc gagcaccacc
     1621 accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     1681 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     1741 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
     1801 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     1861 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     1921 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     1981 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     2041 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     2101 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     2161 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     2221 attaacgttt acaatttcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     2281 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     2341 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     2401 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     2461 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     2521 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     2581 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     2641 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     2701 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     2761 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     2821 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     2881 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     2941 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     3001 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     3061 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     3121 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     3181 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3241 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3301 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3361 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     3421 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3481 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3541 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3601 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3661 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3721 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3781 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3841 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3901 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     3961 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4021 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4081 tcacaccgca tatatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     4141 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     4201 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4261 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4321 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     4381 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     4441 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     4501 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     4561 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     4621 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     4681 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     4741 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     4801 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     4861 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     4921 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     4981 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     5041 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     5101 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     5161 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     5221 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     5281 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5341 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     5401 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     5461 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     5521 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     5581 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     5641 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     5701 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     5761 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     5821 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     5881 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     5941 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     6001 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     6061 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     6121 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     6181 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     6241 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     6301 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     6361 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     6421 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     6481 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     6541 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     6601 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     6661 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     6721 gcgtagagga tcg
//
