LOCUS       sequence-213526-        7319 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Funclib design ACS_19.
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 Dec 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_19
FEATURES             Location/Qualifiers
     source          1..7319
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     6..25
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     109..128
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        109..127
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    128..152
                     /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             167..189
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2159..2176
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2229..2247)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2243..2290
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2327..2782
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2414..2433)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2624..2645
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2808..2912
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2913..2981,2982..3773)
                     /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(3131..3150)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3944..4532
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4433..4452
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4686..4703
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4718..4857
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4783..4802)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4943..4965
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4959..5150)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHT
                     DELYRSCLARFGDDGENL"
     CDS             complement(5959..7041)
                     /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     7003..7022
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7042..7119)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
       61 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca
      121 ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta
      181 agaaggagat atacatatgt ctcagaccca caaacacccg gttccgccga acatcgcggc
      241 gaacgcgtgg atcaacccgg aacagtacga agcgatgtac aaacagtcta tcaacgaccc
      301 ggacaccttc tggcgtgaac agggtaaaat cctggactgg atcaaaccgt acacccgtgt
      361 taaaaacacc tctttcgaac cgggtaacgt ttctatccgt tggtacgaag acggtaccct
      421 gaacctggcg tacaactgcc tggaccgtca cctggaagaa cgtggtgacc agaccgcgat
      481 catctgggaa ggtgacgacc cgtctgaatc tcgtcacatc acctaccgtg aactgcaccg
      541 tgacgtttgc cgtttcgcga acgttctgaa agcgctgggt atcaaaaaag gtgacgttgt
      601 tgcgatctac atgccgatgg ttccggaagc ggcggttgcg atgctggcgt gcgcgcgtat
      661 cggtgcgatc cactctgtta tcttcggtgg tttctctccg gaagcggttg cgggtcgtat
      721 catcgactct aactctcgtc tggttatcac cgcggacgaa ggtgttcgtg cgggtcgttc
      781 tatcccgctg aaaaagaacg ttgacgaagc gctgaaaaac ccgaacgtta cctctgttga
      841 acacgttatc gttttcaaac gtaccggtgg tgacatcgac tggcaggaag gtcgtgacct
      901 gtggtggcgt gacctgatcg aaaaagcgtc tccggaacac cagccggaac cgatgaacgc
      961 ggaagacccg ctgttcatcc tgtacacctc tggttctacc ggtaaaccga aaggtgttct
     1021 gcacaccacc ggtggttacc tggtttacgc ggcgaccacc ttcaaatacg ttttcgacta
     1081 ccacccgggt gacatctact ggtgcaccgc ggacgttggt tggattaccg gtcacgcgta
     1141 tctgctgtac ggtccgctgg cgtgcggtgc gaccaccctg atgttcgaag gtgttccgaa
     1201 ctggccgacc ccgtctcgta tggcgcaggt tgttgacaaa caccaggtta acatcctgtt
     1261 taccgcgccg accgcgatcc gtgcgctgat ggcggaaggt gacaaagcga tcgaaggtac
     1321 cgaccgttct tctctgcgta tcctgggttc taccggtgaa ccgatcaacc cggaagcgtg
     1381 ggaatggtac tggaaaaaaa tcggtaacga acgttgcccg gttgttgaca cctggtggca
     1441 gaccgaaacc ggtggttgca tgatcacccc gctgccgggt gcgatcgaac tgaaaccggg
     1501 ttctgcgacc cgtccgttct tcggtgttca gccggcgctg gttgacaacg aaggtaaccc
     1561 gcaggaaggt gcgaccgaag gtaacctggt tatcaccgac tcttggccgg gtcaggcgcg
     1621 taccctgtac ggtgaccacg aacgtttcga acagacctac ttctctacct tcaaaggtat
     1681 gtacttctct ggtgacggtg cgcgtcgtga cgaagacggt tactactgga tcaccggtcg
     1741 tgttgacgac gttctgaacg tttctggtca ccgtctgggt accgcggaaa tcgaatctgc
     1801 gctggttgcg cacccgaaaa tcgcggaagc ggcggttgtt ggtatcccgc acgacatcaa
     1861 aggtcaggcg atctacgcgt acgttaccct gaaccacggt gaagaaccgt ctccggaact
     1921 gtacaaagaa gttcgtaact gggttcgtaa agaaatcggt ccgctggcga ccccggacgt
     1981 tctgcactgg accgacggtc tgccgaaaac ccgttctggt aaaatcatgc gtcgtatcct
     2041 gcgtaaaatc gcggcgggtg acacctctaa cctgggtgac acctctaccc tggcggaccc
     2101 gtctgttgtt gacaaactga tcgaagaaaa acaggcgatc gcgatgccgt ctctcgagca
     2161 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     2221 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     2281 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
     2341 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     2401 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     2461 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     2521 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     2581 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     2641 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     2701 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     2761 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     2821 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     2881 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     2941 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     3001 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     3061 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     3121 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     3181 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     3241 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     3301 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     3361 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     3421 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     3481 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     3541 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     3601 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     3661 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     3721 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     3781 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     3841 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     3901 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3961 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     4021 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     4081 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4141 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4201 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4261 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4321 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4381 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4441 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4501 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4561 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     4621 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     4681 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     4741 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     4801 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     4861 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     4921 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4981 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagtgt ggtcgtgaag cgattcacag
     5041 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     5101 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     5161 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     5221 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     5281 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     5341 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     5401 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     5461 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     5521 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     5581 gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg
     5641 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     5701 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     5761 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     5821 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     5881 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     5941 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     6001 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     6061 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     6121 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     6181 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     6241 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     6301 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     6361 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     6421 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     6481 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     6541 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     6601 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     6661 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     6721 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     6781 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     6841 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     6901 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     6961 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     7021 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     7081 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     7141 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     7201 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     7261 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttcc
//
