LOCUS       sequence-489559-        5674 bp    DNA     circular SYN 16-OCT-2025
DEFINITION  GGA cloning MSG-AGGA-promoter-ccdb-TTCC-HIS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5674)
  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 5674)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 16, 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: cwby0002
FEATURES             Location/Qualifiers
     source          1..5674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      15..470
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(102..121)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     312..333
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(563..1378)
                     /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"
     CDS             complement(563..1378)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1286..1305
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1370..1389
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1500..2088
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1989..2008
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2242..2259
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2274..2416
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2342..2361)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2502..2524
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2518..2709)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3518..4600)
                     /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     4562..4581
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4601..4678)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4884..4903
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4991..5010
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4991..5009
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5010..5034
                     /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             5049..5071
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5199..5504
                     /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     5383..5402
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             5518..5535
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5591..5609)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5605..5652
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
       61 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
      121 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
      181 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
      241 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
      301 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
      361 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
      421 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt caggtggcac
      481 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
      541 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
      601 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
      661 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
      721 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
      781 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
      841 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
      901 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
      961 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     1021 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     1081 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     1141 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     1201 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     1261 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     1321 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     1381 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     1441 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     1501 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     1561 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     1621 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     1681 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     1741 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     1801 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     1861 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     1921 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     1981 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     2041 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     2101 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     2161 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     2221 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     2281 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg tgcactctca
     2341 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     2401 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     2461 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     2521 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     2581 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     2641 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     2701 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     2761 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     2821 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     2881 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     2941 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     3001 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     3061 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     3121 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc
     3181 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     3241 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     3301 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     3361 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     3421 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     3481 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     3541 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     3601 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     3661 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     3721 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     3781 cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     3841 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     3901 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     3961 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     4021 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     4081 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     4141 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     4201 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     4261 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     4321 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     4381 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     4441 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     4501 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     4561 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     4621 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     4681 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     4741 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     4801 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     4861 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
     4921 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat cgatctcgat
     4981 cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta
     5041 gaaataattt tgtttaactt taagaaggag atatacatat gtcaggagga gaccgaaagc
     5101 gtattacagt gacagttgac agcgacagct atcagttgct caaggcatat atgatgtcaa
     5161 tatctccggt ctggtatgca caaccaagaa tgaagcccat gcagtttaag gtttacacct
     5221 ataaaagaga gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc
     5281 ccgggcgacg gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc
     5341 gtgaacttta cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata
     5401 tggccagtgt gccggtttcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa
     5461 atgacatcaa aaacgccatt aacctgatgt tctggggaat atgaacggtc tcgttcccac
     5521 caccaccacc accactaatg agatccggct gctaacaaag cccgaaagga agctgagttg
     5581 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     5641 aggggttttt tgctgaaagg aggaactata tccg
//
