LOCUS       sequence-213487-        7319 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Funclib design ACS_4.
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_4
FEATURES             Location/Qualifiers
     source          1..7319
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     36..55
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     139..158
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        139..157
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    158..182
                     /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             197..219
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2189..2206
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2259..2277)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2273..2320
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2357..2812
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2444..2463)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2654..2675
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2838..2942
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2943..3011,3012..3803)
                     /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(3161..3180)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3974..4562
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4463..4482
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4716..4733
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4748..4887
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4813..4832)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4973..4995
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4989..5180)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5989..7071)
                     /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     7033..7052
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7072..7149)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
       61 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
      121 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
      181 cccctctaga aataattttg tttaacttta agaaggagat atacatatgt ctcagaccca
      241 caaacacccg gttccgccga acatcgcggc gaacgcgtgg atcaacccgg aacagtacga
      301 agcgatgtac aaacagtcta tcaacgaccc ggacaccttc tggcgtgaac agggtaaaat
      361 cctggactgg atcaaaccgt acacccgtgt taaaaacacc tctttcgaac cgggtaacgt
      421 ttctatccgt tggtacgaag acggtaccct gaacctggcg tacaactgcc tggaccgtca
      481 cctggaagaa cgtggtgacc agaccgcgat catctgggaa ggtgacgacc cgtctgaatc
      541 tcgtcacatc acctaccgtg aactgcaccg tgacgtttgc cgtttcgcga acgttctgaa
      601 agcgctgggt atcaaaaaag gtgacgttgt tgcgatctac atgccgatgg ttccggaagc
      661 ggcggttgcg atgctggcgt gcgcgcgtat cggtgcgatc cactctgtta tcttcggtgg
      721 tttctctccg gaagcggttg cgggtcgtat catcgactct aactctcgtc tggttatcac
      781 cgcggacgaa ggtgttcgtg cgggtcgttc tatcccgctg aaaaagaacg ttgacgaagc
      841 gctgaaaaac ccgaacgtta cctctgttga acacgttatc gttttcaaac gtaccggtgg
      901 tgacatcgac tggcaggaag gtcgtgacct gtggtggcgt gacctgatcg aaaaagcgtc
      961 tccggaacac cagccggaac cgatgaacgc ggaagacccg ctgttcatcc tgtacacctc
     1021 tggttctacc ggtaaaccga aaggtgttct gcacaccacc ggtggttacc tggtttacgc
     1081 ggcgaccacc ttcaaatacg ttttcgacta ccacccgggt gacatctact ggtgcaccgc
     1141 ggacgttggt tgggcggtgg gtcacaccta tctgctgtac ggtccgctgg cgtgcggtgc
     1201 gaccaccctg atgttcgaag gtgttccgaa ctggccgacc ccgtctcgta tggcgcaggt
     1261 tgttgacaaa caccaggtta acatcctgtt taccgcgccg accgcgatcc gtgcgctgat
     1321 ggcggaaggt gacaaagcga tcgaaggtac cgaccgttct tctctgcgta tcgtgggttc
     1381 taccggtgaa ccgatcaacc cggaagcgtg ggaatggtac tggaaaaaaa tcggtaacga
     1441 acgttgcccg gttgttgaca cctggtggca gaccgaaacc ggtggtgcga tgatcacccc
     1501 gctgccgggt gcgatcgaac tgaaaccggg ttctgcgacc cgtccgttct tcggtgttca
     1561 gccggcgctg gttgacaacg aaggtaaccc gcaggaaggt gcgaccgaag gtaacctggt
     1621 tatcaccgac tcttggccgg gtcaggcgcg taccctgtac ggtgaccacg aacgtttcga
     1681 acagacctac ttctctacct tcaaaggtat gtacttctct ggtgacggtg cgcgtcgtga
     1741 cgaagacggt tactactgga tcaccggtcg tgttgacgac gttctgaacg tttctggtca
     1801 ccgtctgggt accgcggaaa tcgaatctgc gctggttgcg cacccgaaaa tcgcggaagc
     1861 ggcggttgtt ggtatcccgc acgacatcaa aggtcaggcg atctacgcgt acgttaccct
     1921 gaaccacggt gaagaaccgt ctccggaact gtacaaagaa gttcgtaact gggttcgtaa
     1981 agaaatcggt ccgctggcga ccccggacgt tctgcactgg accgacggtc tgccgaaaac
     2041 ccgttctggt aaaatcatgc gtcgtatcct gcgtaaaatc gcggcgggtg acacctctaa
     2101 cctgggtgac acctctaccc tggcggaccc gtctgttgtt gacaaactga tcgaagaaaa
     2161 acaggcgatc gcgatgccgt ctctcgagca ccaccaccac caccactgag atccggctgc
     2221 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     2281 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     2341 cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     2401 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     2461 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     2521 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     2581 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     2641 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     2701 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     2761 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     2821 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     2881 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     2941 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     3001 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     3061 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     3121 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     3181 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     3241 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     3301 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     3361 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     3421 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     3481 tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     3541 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     3601 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     3661 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     3721 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     3781 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     3841 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     3901 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     3961 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4021 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4081 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     4141 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4201 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4261 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4321 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4381 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4441 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4501 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4561 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     4621 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     4681 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     4741 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     4801 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     4861 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     4921 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     4981 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     5041 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     5101 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     5161 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     5221 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     5281 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     5341 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     5401 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     5461 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     5521 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     5581 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     5641 acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     5701 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     5761 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     5821 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     5881 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     5941 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     6001 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     6061 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     6121 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     6181 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     6241 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     6301 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     6361 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     6421 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     6481 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     6541 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     6601 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     6661 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     6721 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     6781 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     6841 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     6901 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     6961 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     7021 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     7081 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     7141 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     7201 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     7261 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctc
//
