LOCUS       sequence-494299-        5839 bp    DNA     circular SYN 02-DEC-2025
DEFINITION  Demultiplexing 
            M-BsaI_AGGA_BsmBI-promoter-ccdb-BsmBI_TTCC_BsaI_his-W__AMPR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5839)
  AUTHORS   Qian J, Milles LF, Wicky BIM, Motmaen A, Li X, Kibler RD, Stewart L,
            Baker D
  TITLE     Accelerating protein design by scaling experimental characterization
  JOURNAL   bioRxiv 2025.08.05.668824; doi: 
            https://doi.org/10.1101/2025.08.05.668824
REFERENCE   2  (bases 1 to 5839)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 2, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2025.08.05.668824; doi: https://doi.org/10.1101/2025.08.05.668824"
COMMENT     Sequence Label: DMX0001
FEATURES             Location/Qualifiers
     source          1..5839
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     157..176
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     264..283
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        264..282
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    283..307
                     /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             322..344
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             478..783
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     662..681
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             803..820
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(879..897)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      893..940
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      977..1432
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1064..1083)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1274..1295
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1525..2316,2317..2385))
                     /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"
     primer_bind     2148..2167
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2386..2490)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2612..3200
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3101..3120
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3354..3371
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3386..3528
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3454..3473)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3614..3636
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3630..3821)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4630..5712)
                     /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     5674..5693
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5713..5790)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
       61 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
      121 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
      181 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
      241 gatcgatctc gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa
      301 caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatataca tatggggtct
      361 caaggaggag acggaaagcg tattacagtg acagttgaca gcgacagcta tcagttgctc
      421 aaggcatata tgatgtcaat atctccggtc tggtatgcac aaccaagaat gaagcccatg
      481 cagtttaagg tttacaccta taaaagagag agccgttatc gtctgtttgt ggatgtacag
      541 agtgatatta ttgacacgcc cgggcgacgg atggtgatcc ccctggccag tgcacgtctg
      601 ctgtcagata aagtctcccg tgaactttac ccggtggtgc atatcgggga tgaaagctgg
      661 cgcatgatga ccaccgatat ggccagtgtg ccggtttccg ttatcgggga agaagtggct
      721 gatctcagcc accgcgaaaa tgacatcaaa aacgccatta acctgatgtt ctggggaata
      781 tgaaccgtct cgttccggag accaccacca ccaccaccac tggtaatgag atccggctgc
      841 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
      901 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
      961 cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     1021 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     1081 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     1141 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     1201 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     1261 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     1321 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     1381 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat ttcaggtggc
     1441 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     1501 atgtatccgc tcatgaatta attcttacca atgcttaatc agtgaggcac ctatctcagc
     1561 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     1621 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     1681 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     1741 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     1801 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     1861 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     1921 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     1981 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     2041 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     2101 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     2161 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     2221 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     2281 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     2341 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     2401 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     2461 ttagaaaaat aaacaaatag gggttccgcg aacacccctt gtattactgt ttatgtaagc
     2521 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2581 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2641 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2701 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     2761 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     2821 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     2881 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     2941 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3001 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3061 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3121 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3181 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3241 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3301 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3361 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     3421 gtatttcaca ccgcatatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     3481 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     3541 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     3601 gctgtgaccg tcaccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     3661 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     3721 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     3781 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     3841 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     3901 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     3961 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     4021 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     4081 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     4141 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     4201 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     4261 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc
     4321 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     4381 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     4441 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     4501 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     4561 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     4621 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4681 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     4741 caccagtgaa acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     4801 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     4861 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc
     4921 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     4981 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     5041 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     5101 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     5161 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     5221 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     5281 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     5341 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     5401 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     5461 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     5521 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     5581 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     5641 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     5701 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     5761 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     5821 cctgcattag gaagcagcc
//
