LOCUS       Exported                7118 bp ds-DNA     circular SYN 19-JUN-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7118)
  AUTHORS   Max Perutz Labs
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Jul 8, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7118
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    3..27
                     /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)."
     misc_binding    3..25
                     /locus_tag="LacO"
                     /label=LacO
     RBS             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     misc_feature    71..73
                     /label=ATG
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             113..133
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             134..838
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     854..892
                     /label=SMP5312
     CDS             869..1849
                     /codon_start=1
                     /label=ATG13-IDR (191-517)
                     /note="ATG13"
                     /translation="INLAFMSTRQFERTPPIMGIIIDHFVDRPYPSSSPMHPCNYRTAG
                     EDTGVIYPSVEDSQEVCTTSFSTSPPSQLSSSRLSYQPAALGVGSADLAYPVVFAAGLN
                     ATHPHQLMVPGKEGGVPLAPNQPVHGTQADQERLATCTPSDRTHCAATPSSSEDTETVS
                     NSSEGRASPHDVLETIFVRKVGAFVNKPINQVTLTSLDIPFAMFAPKNLELEDTDPMVN
                     PPDSPETESPLQGSLHSDGSSGGSSGNTHDDFVMIDFKPAFSKDDILPMDLGTFYREFQ
                     NPPQLSSLSIDIGAQSMAEDLDSLPEKLAVHEKNVREFDAFVETLQ"
     primer_bind     complement(869..905)
                     /label=SMP5068
                     /note="13_IDR_3A. can be used with full length and IDR only
                     construct"
     primer_bind     869..893
                     /label=Fragment.FORa
     primer_bind     888..937
                     /label=SMP5067
                     /note="13_IDR_3A. can be used with full length and IDR only
                     construct"
     primer_bind     888..916
                     /label=SMP5069
                     /note="13_IDR_3A. can only be used with full length 
                     construct"
     primer_bind     950..967
                     /label=SMP2547
     primer_bind     complement(1087..1103)
                     /label=SMP2554
     primer_bind     complement(1831..1872)
                     /label=SMP5313
     misc_feature    1889..1906
                     /locus_tag="pET Downstream"
                     /label=pET Downstream
     primer_bind     1912..1931
                     /locus_tag="T7"
                     /label=T7
     promoter        1912..1930
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1931..1955
                     /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)."
     misc_binding    1931..1953
                     /locus_tag="LacO(1)"
                     /label=LacO
                     /label=LacO(1)
     CDS             2064..2108
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     terminator      2160..2207
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2244..2699
                     /locus_tag="M13 origin"
                     /label=M13 origin
     rep_origin      2261..2567
                     /locus_tag="F1 ori"
                     /label=F1 ori
     CDS             complement(2814..3674)
                     /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"
     CDS             complement(2817..3476)
                     /locus_tag="AmpR"
                     /label=AmpR
     promoter        complement(3675..3767)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3772..4454
                     /locus_tag="ColE1 origin"
                     /label=ColE1 origin
     rep_origin      3848..4436
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    4622..4764
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4866..5057)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5629..6711)
                     /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"
     CDS             complement(5632..6588)
                     /locus_tag="LacI"
                     /label=LacI
     primer_bind     6577..6587
                     /label=R134A-E135A_WIPI2d_rv1
                     /note="R134A-E135A_WIPI2d_rv1"
     primer_bind     6577..6587
                     /label=SMP5108
                     /note="R134A-E135A_WIPI2d_rv1"
     promoter        complement(6712..6789)
                     /gene="lacI"
                     /label=lacI promoter
     misc_feature    7056..7070
                     /locus_tag="pET Upstream"
                     /label=pET Upstream
     primer_bind     join(7102..7118,1..3)
                     /locus_tag="T7(1)"
                     /label=T7
                     /label=T7(1)
     promoter        join(7102..7118,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cggaaaacct
      121 gtattttcag ggagtgagca agggcgagga ggataacatg gccatcatca aggagttcat
      181 gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
      241 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg
      301 ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta
      361 cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa
      421 gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc
      481 cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga
      541 cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc
      601 cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca
      661 ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc
      721 ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga
      781 acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagtc
      841 cggactcaga tctcgagctc aagcttcgat caatctggcc ttcatgtcaa ccagacagtt
      901 tgagcgtact ccccctatta tgggaatcat tatcgaccac ttcgtggata gaccataccc
      961 cagtagctcc cctatgcatc catgcaacta tcgtacagcc ggtgaagaca ccggcgtgat
     1021 ctaccccagt gtcgaggata gccaggaagt gtgtaccact tccttctcta cctcaccacc
     1081 ctcacagctc tcttcaagtc gcctgagtta tcagcctgcc gctctcggag tcggtagcgc
     1141 agacctggcc tacccagtgg tcttcgcagc aggcctcaac gcaacccacc cacatcagct
     1201 gatggtgcca ggaaaggagg gtggagtccc actggctcct aatcagccag tgcacggtac
     1261 tcaggctgac caggaaaggc tggcaacttg cacaccatcc gatagaactc attgtgctgc
     1321 aacacccagc tcctctgagg acaccgaaac tgtgagcaac tcaagtgagg gtcgcgcctc
     1381 cccacacgat gtcctggaaa ccatcttcgt gagaaaagtc ggcgcttttg tgaacaagcc
     1441 cattaatcag gtcacactca cctctctgga catccctttc gctatgtttg caccaaagaa
     1501 cctcgagctg gaagacactg atcccatggt gaatcctcca gactcacccg agacagaaag
     1561 tcctctccag ggcagtctgc acagcgatgg aagctccggt ggctcttcag gcaacacaca
     1621 tgacgatttc gtcatgattg acttcaaacc cgctttttcc aaggacgata tcctccctat
     1681 ggatctggga accttctaca gggagtttca gaatccccct cagctcagta gcctgagcat
     1741 tgacatcggt gcccagtcca tggctgagga cctcgattct ctgcctgaaa aactggctgt
     1801 gcacgagaag aacgtcagag aattcgacgc atttgtggag accctgcagt gagcataatg
     1861 cttaagtcga acagaaagta atcgtattgt acacggccgc ataatcgaaa ttaatacgac
     1921 tcactatagg ggaattgtga gcggataaca attccccatc ttagtatatt agttaagtat
     1981 aagaaggaga tatacatatg gcagatctca attggatatc ggccggccac gcgatcgctg
     2041 acgtcggtac cctcgagtct ggtaaagaaa ccgctgctgc gaaatttgaa cgccagcaca
     2101 tggactcgtc tactagcgca gcttaattaa cctaggctgc tgccaccgct gagcaataac
     2161 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     2221 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2281 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2341 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2401 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2461 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2521 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2581 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2641 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc
     2701 tggcggcacg atggcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     2761 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     2821 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     2881 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     2941 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     3001 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     3061 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     3121 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     3181 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     3241 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     3301 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     3361 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     3421 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     3481 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     3541 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     3601 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     3661 tgttgaatac tcatactctt cctttttcaa tcatgattga agcatttatc agggttattg
     3721 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gtcatgacca
     3781 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     3841 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     3901 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     3961 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     4021 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4081 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4141 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4201 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     4261 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     4321 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     4381 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     4441 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     4501 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     4561 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     4621 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatatggtg
     4681 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     4741 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     4801 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4861 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     4921 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     4981 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     5041 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     5101 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     5161 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     5221 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     5281 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     5341 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     5401 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     5461 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgctagtc
     5521 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     5581 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     5641 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5701 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     5761 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     5821 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     5881 ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt
     5941 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     6001 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     6061 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     6121 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     6181 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     6241 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     6301 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     6361 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     6421 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     6481 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     6541 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     6601 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     6661 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     6721 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     6781 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     6841 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     6901 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     6961 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     7021 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     7081 tcgatctcga tcccgcgaaa ttaatacgac tcactata
//
