LOCUS       sequence-346213-        4542 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  yeast MoClo level-0 part plasmid type 6 containing non-coding DNA 
            spacer for construction of MoClo plasmids without yeast marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4542)
  AUTHORS   Otto M, Skrekas C, Gossing M, Gustafsson J, Siewers V, David F
  TITLE     Expansion of the Yeast Modular Cloning Toolkit for CRISPR-Based 
            Applications, Genomic Integrations and Combinatorial Libraries.
  JOURNAL   ACS Synth Biol. 2021 Dec 17;10(12):3461-3474. doi: 
            10.1021/acssynbio.1c00408. Epub 2021 Dec 3.
  PUBMED    34860007
REFERENCE   2  (bases 1 to 4542)
  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: 
            "10.1021/acssynbio.1c00408"; journalName: "ACS Synth Biol"; date: 
            "2021-12-17- 17"; volume: "10"; issue: "12"; pages: "3461-3474"
COMMENT     Sequence Label: pMC6-spacer
FEATURES             Location/Qualifiers
     source          1..4542
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     236..255
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(474..565)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(606..653)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     649..667
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(724..741)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1675..1697
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1712..1736
                     /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)."
     primer_bind     complement(1736..1755)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1737..1755)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1839..1858)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2064..2141
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2142..3224
                     /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     complement(2161..2180)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4033..4224
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4218..4240)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4326..4465
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4381..4400
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4480..4497)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
       61 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
      121 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
      181 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
      241 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
      301 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
      361 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
      421 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
      481 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
      541 gaatgtattt agaaaaataa acaaataggc ccattcgcca atccggatat agttcctcct
      601 ttcagcaaaa aacccctcaa gacccgttta gaggccccaa ggggttatgc tagttattgc
      661 tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt tgttagcagc cggatcgctg
      721 ttagtggtgg tgatgatgat gtcctccttc cttgcgcgcc tgacgggcaa tctcctccgc
      781 ctggcgggct gcttcctcag caaggcgcgc tgctttctca agcagttctt catttccgcg
      841 atcgcgcgca atttctgcca gaacatatgc agcgttgacc aggatttgaa gggcctttaa
      901 tgcaagctcc aagttgcctt ctttacgagc ttggcgcgca atttcttctg cctgacgcgc
      961 agcttcctcc gcaagacgtg ccgctttctc taaaagctcc tcattgccgc ggtcacgggc
     1021 gatctcagca aggacgtacg ccgcgtttac taaaatctgc aaagctttca gcgccaactc
     1081 taaattgcct tccttacgag cctgacgcgc gatttcttcc gcttgacggg cagcttcttc
     1141 agcaagacgc gccgccttct ccaacaattc ttcgttgcca cgatcgcgtg caatctcagc
     1201 taatacataa gcagcattca caagaatttg cagtgctttc aatgcaagtt caagattacc
     1261 tccaggtccc ccaaggaact caggtgcgct tgacactgtt acttgagtgc cctgtcccca
     1321 atagtcgtag tcgtaatccc actctgatac tactgttcct aaccccgctg ccgcgcaata
     1381 atatactgct gtatcgtcgg gcttcaggct attcatttgc aagtagacgg tgtttttcgc
     1441 gttgtcgcgg gagatagtga aacgtccttt tactgaacgg gtgtaataag tagagccacc
     1501 actccatgag atagttgcaa caaattcacg ttccttacca ggggcctggc gaaaccaacc
     1561 cattgcgtac tcactaaagg tacggccaga ggcggcacag gacaaacgaa ggctgccgcc
     1621 cgcctgaacc aacccaccgc cgctttcttg aagttgtact tgtcccatag tatatctcct
     1681 tcttaaagtt aaacaaaatt atttctagag gggaattgtt atccgctcac aattccccta
     1741 tagtgagtcg tattaatttc gcgggatcga gatctcgatc ctctacgccg gacgcatcgt
     1801 ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg acatcaccga
     1861 tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg tgggtatggt
     1921 ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcac cattccttgc
     1981 ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca
     2041 taagggagag cgtcgagatc ccggacacca tcgaatggcg caaaaccttt cgcggtatgg
     2101 catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat
     2161 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg
     2221 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt
     2281 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg
     2341 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg
     2401 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct
     2461 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc
     2521 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat
     2581 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaagacggta
     2641 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg
     2701 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc
     2761 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc
     2821 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg
     2881 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg
     2941 atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa
     3001 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac
     3061 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa
     3121 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     3181 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     3241 gtaagttagc tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac
     3301 ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc
     3361 ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag
     3421 gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg
     3481 cacgccctcg ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag
     3541 gccattatcg ccggcatggc ggccccacgg gtgcgcatga tcgtgctcct gtcgttgagg
     3601 acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag
     3661 cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc
     3721 ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg
     3781 ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta
     3841 acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc
     3901 agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc
     3961 gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac
     4021 acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga
     4081 agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac
     4141 atctgtgaat cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc
     4201 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     4261 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     4321 cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg
     4381 cggcatcaga gcagattgta ctgagagtgc accattgcgg tgtgaaatac cgcacagatg
     4441 cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4501 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aa
//
