LOCUS       sequence-213497-        7319 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Funclib design ACS_16.
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_16
FEATURES             Location/Qualifiers
     source          1..7319
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     47..66
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     150..169
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        150..168
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    169..193
                     /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             208..230
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2200..2217
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2270..2288)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2284..2331
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2368..2823
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2455..2474)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2665..2686
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2849..2953
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2954..3022,3023..3814)
                     /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(3172..3191)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3985..4573
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4474..4493
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4727..4744
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4759..4898
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4824..4843)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4984..5006
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5000..5191)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6000..7082)
                     /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     7044..7063
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7083..7160)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
       61 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
      121 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
      181 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg
      241 tctcagaccc acaaacaccc ggttccgccg aacatcgcgg cgaacgcgtg gatcaacccg
      301 gaacagtacg aagcgatgta caaacagtct atcaacgacc cggacacctt ctggcgtgaa
      361 cagggtaaaa tcctggactg gatcaaaccg tacacccgtg ttaaaaacac ctctttcgaa
      421 ccgggtaacg tttctatccg ttggtacgaa gacggtaccc tgaacctggc gtacaactgc
      481 ctggaccgtc acctggaaga acgtggtgac cagaccgcga tcatctggga aggtgacgac
      541 ccgtctgaat ctcgtcacat cacctaccgt gaactgcacc gtgacgtttg ccgtttcgcg
      601 aacgttctga aagcgctggg tatcaaaaaa ggtgacgttg ttgcgatcta catgccgatg
      661 gttccggaag cggcggttgc gatgctggcg tgcgcgcgta tcggtgcgat ccactctgtt
      721 atcttcggtg gtttctctcc ggaagcggtt gcgggtcgta tcatcgactc taactctcgt
      781 ctggttatca ccgcggacga aggtgttcgt gcgggtcgtt ctatcccgct gaaaaagaac
      841 gttgacgaag cgctgaaaaa cccgaacgtt acctctgttg aacacgttat cgttttcaaa
      901 cgtaccggtg gtgacatcga ctggcaggaa ggtcgtgacc tgtggtggcg tgacctgatc
      961 gaaaaagcgt ctccggaaca ccagccggaa ccgatgaacg cggaagaccc gctgttcatc
     1021 ctgtacacct ctggttctac cggtaaaccg aaaggtgttc tgcacaccac cggtggttac
     1081 ctggtttacg cggcgaccac cttcaaatac gttttcgact accacccggg tgacatctac
     1141 tggtgcaccg cggacgttgg ttggattgtg ggtcacacct atctgctgta cggtccgctg
     1201 gcgtgcggtg cgaccaccct gatgttcgaa ggtgttccga actggccgac cccgtctcgt
     1261 atggcgcagg ttgttgacaa acaccaggtt aacatcctgt ttaccgcgcc gaccgcgatc
     1321 cgtgcgctga tggcggaagg tgacaaagcg atcgaaggta ccgaccgttc ttctctgcgt
     1381 atcctgggtt ctctgggtga accgatcaac ccggaagcgt gggaatggta ctggaaaaaa
     1441 atcggtaacg aacgttgccc ggttgttgac acctggtggc agaccgaaac cggtggtgcg
     1501 atgatcaccc cgctgccggg tgcgatcgaa ctgaaaccgg gttctgcgac ccgtccgttc
     1561 ttcggtgttc agccggcgct ggttgacaac gaaggtaacc cgcaggaagg tgcgaccgaa
     1621 ggtaacctgg ttatcaccga ctcttggccg ggtcaggcgc gtaccctgta cggtgaccac
     1681 gaacgtttcg aacagaccta cttctctacc ttcaaaggta tgtacttctc tggtgacggt
     1741 gcgcgtcgtg acgaagacgg ttactactgg atcaccggtc gtgttgacga cgttctgaac
     1801 gtttctggtc accgtctggg taccgcggaa atcgaatctg cgctggttgc gcacccgaaa
     1861 atcgcggaag cggcggttgt tggtatcccg cacgacatca aaggtcaggc gatctacgcg
     1921 tacgttaccc tgaaccacgg tgaagaaccg tctccggaac tgtacaaaga agttcgtaac
     1981 tgggttcgta aagaaatcgg tccgctggcg accccggacg ttctgcactg gaccgacggt
     2041 ctgccgaaaa cccgttctgg taaaatcatg cgtcgtatcc tgcgtaaaat cgcggcgggt
     2101 gacacctcta acctgggtga cacctctacc ctggcggacc cgtctgttgt tgacaaactg
     2161 atcgaagaaa aacaggcgat cgcgatgccg tctctcgagc accaccacca ccaccactga
     2221 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     2281 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     2341 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     2401 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     2461 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     2521 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     2581 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     2641 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     2701 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     2761 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     2821 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     2881 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     2941 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     3001 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     3061 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     3121 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     3181 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     3241 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     3301 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     3361 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     3421 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     3481 tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     3541 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     3601 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     3661 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     3721 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     3781 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     3841 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     3901 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3961 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     4021 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4081 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     4141 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4201 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4261 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4321 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4381 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4441 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4501 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4561 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     4621 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4681 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     4741 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4801 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     4861 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     4921 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4981 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     5041 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     5101 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     5161 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     5221 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     5281 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     5341 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     5401 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     5461 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     5521 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     5581 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     5641 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     5701 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     5761 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     5821 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     5881 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     5941 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     6001 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     6061 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     6121 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     6181 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     6241 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     6301 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     6361 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     6421 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     6481 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     6541 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     6601 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     6661 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     6721 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     6781 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     6841 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     6901 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     6961 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     7021 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     7081 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     7141 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     7201 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     7261 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccga
//
