LOCUS       sequence-213523-        7319 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Funclib design ACS_10.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7319)
  AUTHORS   Khersonsky O, Lipsh R, Avizemer Z, Ashani Y, Goldsmith M, Leader H, 
            Dym O, Rogotner S, Trudeau DL, Prilusky J, Amengual-Rigo P, Guallar 
            V, Tawfik DS, Fleishman SJ
  TITLE     Automated Design of Efficient and Functionally Diverse Enzyme 
            Repertoires.
  JOURNAL   Mol Cell. 2018 Oct 4;72(1):178-186.e5. doi: 
            10.1016/j.molcel.2018.08.033. Epub 2018 Sep 27.
  PUBMED    30270109
REFERENCE   2  (bases 1 to 7319)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2018.08.033"; journalName: "Mol Cell"; date: 
            "2018-10-4- 4"; volume: "72"; issue: "1"; pages: "178-186.e5"
COMMENT     Sequence Label: ACS_10
FEATURES             Location/Qualifiers
     source          1..7319
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     44..63
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     147..166
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        147..165
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    166..190
                     /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             205..227
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2197..2214
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2267..2285)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2281..2328
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2365..2820
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2452..2471)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2662..2683
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2846..2950
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2951..3019,3020..3811)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3169..3188)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3982..4570
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4471..4490
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4724..4741
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4756..4895
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4821..4840)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4981..5003
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4997..5188)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5997..7079)
                     /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
                     EDGTRLGVEHLVALGHQQIALLSGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     7041..7060
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7080..7157)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
       61 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
      121 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
      181 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat acatatgtct
      241 cagacccaca aacacccggt tccgccgaac atcgcggcga acgcgtggat caacccggaa
      301 cagtacgaag cgatgtacaa acagtctatc aacgacccgg acaccttctg gcgtgaacag
      361 ggtaaaatcc tggactggat caaaccgtac acccgtgtta aaaacacctc tttcgaaccg
      421 ggtaacgttt ctatccgttg gtacgaagac ggtaccctga acctggcgta caactgcctg
      481 gaccgtcacc tggaagaacg tggtgaccag accgcgatca tctgggaagg tgacgacccg
      541 tctgaatctc gtcacatcac ctaccgtgaa ctgcaccgtg acgtttgccg tttcgcgaac
      601 gttctgaaag cgctgggtat caaaaaaggt gacgttgttg cgatctacat gccgatggtt
      661 ccggaagcgg cggttgcgat gctggcgtgc gcgcgtatcg gtgcgatcca ctctgttatc
      721 ttcggtggtt tctctccgga agcggttgcg ggtcgtatca tcgactctaa ctctcgtctg
      781 gttatcaccg cggacgaagg tgttcgtgcg ggtcgttcta tcccgctgaa aaagaacgtt
      841 gacgaagcgc tgaaaaaccc gaacgttacc tctgttgaac acgttatcgt tttcaaacgt
      901 accggtggtg acatcgactg gcaggaaggt cgtgacctgt ggtggcgtga cctgatcgaa
      961 aaagcgtctc cggaacacca gccggaaccg atgaacgcgg aagacccgct gttcatcctg
     1021 tacacctctg gttctaccgg taaaccgaaa ggtgttctgc acaccaccgg tggttacctg
     1081 gtttacgcgg cgaccacctt caaatacgtt ttcgactacc acccgggtga catctactgg
     1141 tgcaccgcgg acgttggttg ggcgaccggt cacacctttc tgctgtacgg tccgctggcg
     1201 tgcggtgcga ccaccctgat gttcgaaggt gttccgaact ggccgacccc gtctcgtatg
     1261 gcgcaggttg ttgacaaaca ccaggttaac atcctgtata ccgcgccgac cgcgatccgt
     1321 gcgctgatgg cggaaggtga caaagcgatc gaaggtaccg accgttcttc tctgcgtatc
     1381 ctgggttcta ccggtgaacc gatcaacccg gaagcgtggg aatggtactg gaaaaaaatc
     1441 ggtaacgaac gttgcccggt tgttgacacc tggtggcaga ccgaaaccgg tggtgcgatg
     1501 atcaccccgc tgccgggtgc gatcgaactg aaaccgggtt ctgcgacccg tccgttcttc
     1561 ggtgttcagc cggcgctggt tgacaacgaa ggtaacccgc aggaaggtgc gaccgaaggt
     1621 aacctggtta tcaccgactc ttggccgggt caggcgcgta ccctgtacgg tgaccacgaa
     1681 cgtttcgaac agacctactt ctctaccttc aaaggtatgt acttctctgg tgacggtgcg
     1741 cgtcgtgacg aagacggtta ctactggatc accggtcgtg ttgacgacgt tctgaacgtt
     1801 tctggtcacc gtctgggtac cgcggaaatc gaatctgcgc tggttgcgca cccgaaaatc
     1861 gcggaagcgg cggttgttgg tatcccgcac gacatcaaag gtcaggcgat ctacgcgtac
     1921 gttaccctga accacggtga agaaccgtct ccggaactgt acaaagaagt tcgtaactgg
     1981 gttcgtaaag aaatcggtcc gctggcgacc ccggacgttc tgcactggac cgacggtctg
     2041 ccgaaaaccc gttctggtaa aatcatgcgt cgtatcctgc gtaaaatcgc ggcgggtgac
     2101 acctctaacc tgggtgacac ctctaccctg gcggacccgt ctgttgttga caaactgatc
     2161 gaagaaaaac aggcgatcgc gatgccgtct ctcgagcacc accaccacca ccactgagat
     2221 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     2281 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     2341 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2401 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2461 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2521 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2581 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2641 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2701 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2761 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     2821 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2881 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2941 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3001 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3061 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     3121 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     3181 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3241 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3301 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3361 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3421 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3481 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3541 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3601 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3661 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3721 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3781 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     3841 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     3901 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3961 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4021 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4081 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     4141 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4201 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4261 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4321 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4381 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4441 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4501 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4561 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4621 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4681 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4741 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4801 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     4861 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     4921 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     4981 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     5041 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     5101 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     5161 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     5221 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     5281 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     5341 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     5401 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     5461 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     5521 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     5581 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     5641 tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     5701 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     5761 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     5821 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     5881 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     5941 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     6001 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     6061 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     6121 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     6181 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     6241 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
     6301 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     6361 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     6421 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     6481 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgataacag cgcgatttgc
     6541 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     6601 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     6661 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     6721 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     6781 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     6841 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     6901 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     6961 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     7021 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     7081 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     7141 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     7201 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     7261 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaac
//
