LOCUS       sequence-213524-        7319 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Funclib design ACS_14.
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_14
FEATURES             Location/Qualifiers
     source          1..7319
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1879..1896
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1949..1967)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1963..2010
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2047..2502
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2134..2153)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2344..2365
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2528..2632
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2633..2701,2702..3493)
                     /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(2851..2870)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3664..4252
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4153..4172
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4406..4423
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4438..4577
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4503..4522)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4663..4685
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4679..4870)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5679..6761)
                     /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     6723..6742
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6762..6839)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7045..7064
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7148..7167
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7148..7166
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7167..7191
                     /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             7206..7228
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 aaacagtcta tcaacgaccc ggacaccttc tggcgtgaac agggtaaaat cctggactgg
       61 atcaaaccgt acacccgtgt taaaaacacc tctttcgaac cgggtaacgt ttctatccgt
      121 tggtacgaag acggtaccct gaacctggcg tacaactgcc tggaccgtca cctggaagaa
      181 cgtggtgacc agaccgcgat catctgggaa ggtgacgacc cgtctgaatc tcgtcacatc
      241 acctaccgtg aactgcaccg tgacgtttgc cgtttcgcga acgttctgaa agcgctgggt
      301 atcaaaaaag gtgacgttgt tgcgatctac atgccgatgg ttccggaagc ggcggttgcg
      361 atgctggcgt gcgcgcgtat cggtgcgatc cactctgtta tcttcggtgg tttctctccg
      421 gaagcggttg cgggtcgtat catcgactct aactctcgtc tggttatcac cgcggacgaa
      481 ggtgttcgtg cgggtcgttc tatcccgctg aaaaagaacg ttgacgaagc gctgaaaaac
      541 ccgaacgtta cctctgttga acacgttatc gttttcaaac gtaccggtgg tgacatcgac
      601 tggcaggaag gtcgtgacct gtggtggcgt gacctgatcg aaaaagcgtc tccggaacac
      661 cagccggaac cgatgaacgc ggaagacccg ctgttcatcc tgtacacctc tggttctacc
      721 ggtaaaccga aaggtgttct gcacaccacc ggtggttacc tggtttacgc ggcgaccacc
      781 ttcaaatacg ttttcgacta ccacccgggt gacatctact ggtgcaccgc ggacgttggt
      841 tgggtgagcg gtcacgcgta tctgctgtac ggtccgctgg cgtgcggtgc gaccaccctg
      901 atgttcgaag gtgttccgaa ctggccgacc ccgtctcgta tggcgcaggt tgttgacaaa
      961 caccaggtta acatcctgca taccgcgccg accgcgatcc gtgcgctgat ggcggaaggt
     1021 gacaaagcga tcgaaggtac cgaccgttct tctctgcgta tcctgggttc tgcgggtgaa
     1081 ccgatcaacc cggaagcgtg ggaatggtac tggaaaaaaa tcggtaacga acgttgcccg
     1141 gttgttgaca cctggtggca gaccgaaacc ggtggtgcga tgatcacccc gctgccgggt
     1201 gcgatcgaac tgaaaccggg ttctgcgacc cgtccgttct tcggtgttca gccggcgctg
     1261 gttgacaacg aaggtaaccc gcaggaaggt gcgaccgaag gtaacctggt tatcaccgac
     1321 tcttggccgg gtcaggcgcg taccctgtac ggtgaccacg aacgtttcga acagacctac
     1381 ttctctacct tcaaaggtat gtacttctct ggtgacggtg cgcgtcgtga cgaagacggt
     1441 tactactgga tcaccggtcg tgttgacgac gttctgaacg tttctggtca ccgtctgggt
     1501 accgcggaaa tcgaatctgc gctggttgcg cacccgaaaa tcgcggaagc ggcggttgtt
     1561 ggtatcccgc acgacatcaa aggtcaggcg atctacgcgt acgttaccct gaaccacggt
     1621 gaagaaccgt ctccggaact gtacaaagaa gttcgtaact gggttcgtaa agaaatcggt
     1681 ccgctggcga ccccggacgt tctgcactgg accgacggtc tgccgaaaac ccgttctggt
     1741 aaaatcatgc gtcgtatcct gcgtaaaatc gcggcgggtg acacctctaa cctgggtgac
     1801 acctctaccc tggcggaccc gtctgttgtt gacaaactga tcgaagaaaa acaggcgatc
     1861 gcgatgccgt ctctcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
     1921 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     1981 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     2041 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2101 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     2161 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     2221 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     2281 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     2341 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     2401 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     2461 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     2521 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     2581 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     2641 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     2701 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     2761 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     2821 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     2881 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     2941 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     3001 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     3061 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     3121 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc
     3181 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     3241 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     3301 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     3361 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     3421 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     3481 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     3541 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     3601 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     3661 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     3721 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     3781 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     3841 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     3901 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     3961 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4021 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     4081 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     4141 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     4201 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     4261 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     4321 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     4381 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     4441 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc
     4501 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     4561 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     4621 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     4681 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     4741 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     4801 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     4861 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     4921 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     4981 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     5041 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     5101 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     5161 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     5221 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     5281 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg
     5341 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     5401 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     5461 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     5521 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     5581 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     5641 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5701 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5761 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     5821 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     5881 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     5941 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     6001 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     6061 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     6121 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     6181 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     6241 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     6301 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     6361 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     6421 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     6481 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     6541 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     6601 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     6661 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     6721 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     6781 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     6841 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     6901 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     6961 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     7021 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     7081 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
     7141 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa
     7201 ataattttgt ttaactttaa gaaggagata tacatatgtc tcagacccac aaacacccgg
     7261 ttccgccgaa catcgcggcg aacgcgtgga tcaacccgga acagtacgaa gcgatgtac
//
