LOCUS       sequence-493097-        5842 bp    DNA     circular SYN 18-NOV-2025
DEFINITION  Demultiplexing 
            M-BsaI_AGGA_BsmBI-promoter-ccdb-BsmBI_TTCC_BsaI_W-his__AMPR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5842)
  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 5842)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 18, 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: DMX0002
FEATURES             Location/Qualifiers
     source          1..5842
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     38..57
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(181..228)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     224..242
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(298..315)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(341..646)
                     /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     complement(443..462)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     RBS             780..802
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    817..841
                     /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(841..860)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(842..860)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(948..967)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1173..1250
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1251..2333
                     /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(1270..1289)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3142..3333
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3327..3349)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3435..3577
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3490..3509
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3592..3609)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3763..4351)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3843..3862)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4473..4577
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4578..4646,4647..5438)
                     /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     complement(4796..4815)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(join(5531..5842,1..144))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5668..5689)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
       61 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
      121 ttaatgcgcc gctacagggc gcgtcccatt cgccaatccg gatatagttc ctcctttcag
      181 caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt tatgctagtt attgctcagc
      241 ggtggcagca gccaactcag cttcctttcg ggctttgtta gcagccggat ctcattagtg
      301 gtggtggtgg tggtgccagt ggtctccgga acgagacggt tcatattccc cagaacatca
      361 ggttaatggc gtttttgatg tcattttcgc ggtggctgag atcagccact tcttccccga
      421 taacggaaac cggcacactg gccatatcgg tggtcatcat gcgccagctt tcatccccga
      481 tatgcaccac cgggtaaagt tcacgggaga ctttatctga cagcagacgt gcactggcca
      541 gggggatcac catccgtcgc ccgggcgtgt caataatatc actctgtaca tccacaaaca
      601 gacgataacg gctctctctt ttataggtgt aaaccttaaa ctgcatgggc ttcattcttg
      661 gttgtgcata ccagaccgga gatattgaca tcatatatgc cttgagcaac tgatagctgt
      721 cgctgtcaac tgtcactgta atacgctttc cgtctcctcc ttgagacccc atatgtatat
      781 ctccttctta aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc
      841 ccctatagtg agtcgtatta atttcgcggg atcgagatcg atctcgatcc tctacgccgg
      901 acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga
      961 catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt
     1021 gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc
     1081 attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca
     1141 ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc aaaacctttc
     1201 gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag
     1261 taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg
     1321 tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg
     1381 agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga
     1441 ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta
     1501 aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg
     1561 tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca
     1621 ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc
     1681 cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg
     1741 aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc
     1801 tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat
     1861 atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt
     1921 ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg
     1981 ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg
     2041 ttggtgcgga catctcggta gtgggatacg acgataccga agacagctca tgttatatcc
     2101 cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct
     2161 tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc gtttcactgg
     2221 tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     2281 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     2341 gcaattaatg taagttagct cactcattag gcaccgggat ctcgaccgat gcccttgaga
     2401 gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt
     2461 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt
     2521 ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc
     2581 ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc
     2641 gagaagcagg ccattatcgc cggcatggcg gccccacggg tgcgcatgat cgtgctcctg
     2701 tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg
     2761 atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca
     2821 tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc
     2881 accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca
     2941 tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc
     3001 cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta
     3061 acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat
     3121 cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc
     3181 tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa
     3241 caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg cagctgcctc
     3301 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccggt gacggtcaca
     3361 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     3421 ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc
     3481 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatatgc ggtgtgaaat
     3541 accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac
     3601 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     3661 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     3721 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     3781 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     3841 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     3901 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     3961 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4021 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4081 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4141 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4201 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4261 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4321 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4381 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgaaca ataaaactgt
     4441 ctgcttacat aaacagtaat acaaggggtg ttcgcggaac ccctatttgt ttatttttct
     4501 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     4561 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     4621 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     4681 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     4741 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     4801 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     4861 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     4921 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     4981 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     5041 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     5101 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     5161 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     5221 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     5281 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     5341 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     5401 tcgctgagat aggtgcctca ctgattaagc attggtaaga attaattcat gagcggatac
     5461 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     5521 gtgccacctg aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     5581 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     5641 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     5701 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     5761 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     5821 aaagggagcc cccgatttag ag
//
