LOCUS       sequence-493120-        5892 bp    DNA     circular SYN 18-NOV-2025
DEFINITION  GGA cloning AGGA-promoter-ccdb-TTCC-GS-HALC2_065-SNAC-6xhis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5892)
  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 5892)
  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: cwby0032
FEATURES             Location/Qualifiers
     source          1..5892
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     179..198
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     286..305
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        286..304
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    305..329
                     /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             344..366
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             494..799
                     /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     678..697
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1011..1025
                     /codon_start=1
                     /product="tag for protein cleavage using Ni2+ ion (Dang et 
                     al., 2019)"
                     /label=SNAC tag
                     /translation="GSHHW"
     CDS             1035..1052
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1108..1126)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1122..1169
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1206..1661
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1293..1312)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1503..1524
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1753..2568)
                     /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(1753..2568)
                     /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     2476..2495
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2560..2579
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2690..3278
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3179..3198
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3432..3449
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3464..3603
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3529..3548)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3689..3711
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3705..3896)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4705..5787)
                     /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     5749..5768
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5788..5865)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
       61 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
      121 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
      181 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
      241 tgcgtccggc gtagaggatc gagatcgatc tcgatcccgc gaaattaata cgactcacta
      301 taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga
      361 aggagatata catatgtcag gaggagaccg aaagcgtatt acagtgacag ttgacagcga
      421 cagctatcag ttgctcaagg catatatgat gtcaatatct ccggtctggt atgcacaacc
      481 aagaatgaag cccatgcagt ttaaggttta cacctataaa agagagagcc gttatcgtct
      541 gtttgtggat gtacagagtg atattattga cacgcccggg cgacggatgg tgatccccct
      601 ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat
      661 cggggatgaa agctggcgca tgatgaccac cgatatggcc agtgtgccgg tttccgttat
      721 cggggaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct
      781 gatgttctgg ggaatatgaa cggtctcgtt ccggtagcga agaagaaaag ccgatcgtta
      841 ttgatctgaa caaaaccatt gaacgcgatg gccgcaaagt gaaactggtg cgcgcgacca
      901 ttaccgtgga tccggaaacc aacaccatta ccattgatat tgaatacgag ggcggcccga
      961 ttaccaaaga agatttgctg gaagcgttta aactggcggc gagcaaactg ggatcacatc
     1021 attggggcag cacccaccac caccaccacc actaatgaga tccggctgct aacaaagccc
     1081 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     1141 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     1201 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     1261 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     1321 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     1381 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     1441 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     1501 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     1561 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     1621 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     1681 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     1741 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     1801 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     1861 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     1921 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     1981 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     2041 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     2101 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     2161 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     2221 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     2281 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     2341 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     2401 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     2461 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     2521 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     2581 attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt
     2641 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2701 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2761 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2821 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2881 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2941 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     3001 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     3061 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     3121 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     3181 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     3241 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     3301 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     3361 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     3421 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     3481 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     3541 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     3601 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     3661 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     3721 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     3781 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     3841 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     3901 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     3961 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     4021 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     4081 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     4141 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     4201 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     4261 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     4321 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     4381 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     4441 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     4501 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     4561 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     4621 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     4681 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4741 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     4801 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     4861 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     4921 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     4981 cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     5041 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     5101 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     5161 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     5221 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     5281 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     5341 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     5401 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     5461 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     5521 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     5581 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     5641 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     5701 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     5761 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     5821 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     5881 ctcccttatg cg
//
