LOCUS       sequence-213483-        7319 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Funclib design ACS_27.
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_27
FEATURES             Location/Qualifiers
     source          1..7319
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     42..61
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     145..164
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        145..163
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    164..188
                     /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             203..225
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2195..2212
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2265..2283)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2279..2326
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2363..2818
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2450..2469)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2660..2681
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2844..2948
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2949..3017,3018..3809)
                     /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(3167..3186)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3980..4568
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4469..4488
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4722..4739
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4754..4893
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4819..4838)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4979..5001
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4995..5186)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5995..7077)
                     /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     7039..7058
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7078..7155)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
       61 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
      121 atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata
      181 acaattcccc tctagaaata attttgttta actttaagaa ggagatatac atatgtctca
      241 gacccacaaa cacccggttc cgccgaacat cgcggcgaac gcgtggatca acccggaaca
      301 gtacgaagcg atgtacaaac agtctatcaa cgacccggac accttctggc gtgaacaggg
      361 taaaatcctg gactggatca aaccgtacac ccgtgttaaa aacacctctt tcgaaccggg
      421 taacgtttct atccgttggt acgaagacgg taccctgaac ctggcgtaca actgcctgga
      481 ccgtcacctg gaagaacgtg gtgaccagac cgcgatcatc tgggaaggtg acgacccgtc
      541 tgaatctcgt cacatcacct accgtgaact gcaccgtgac gtttgccgtt tcgcgaacgt
      601 tctgaaagcg ctgggtatca aaaaaggtga cgttgttgcg atctacatgc cgatggttcc
      661 ggaagcggcg gttgcgatgc tggcgtgcgc gcgtatcggt gcgatccact ctgttatctt
      721 cggtggtttc tctccggaag cggttgcggg tcgtatcatc gactctaact ctcgtctggt
      781 tatcaccgcg gacgaaggtg ttcgtgcggg tcgttctatc ccgctgaaaa agaacgttga
      841 cgaagcgctg aaaaacccga acgttacctc tgttgaacac gttatcgttt tcaaacgtac
      901 cggtggtgac atcgactggc aggaaggtcg tgacctgtgg tggcgtgacc tgatcgaaaa
      961 agcgtctccg gaacaccagc cggaaccgat gaacgcggaa gacccgctgt tcatcctgta
     1021 cacctctggt tctaccggta aaccgaaagg tgttctgcac accaccggtg gttacctggt
     1081 ttacgcggcg accaccttca aatacgtttt cgactaccac ccgggtgaca tctactggtg
     1141 caccgcggac gttggttggg tggcgggtca cagctatctg ctgtacggtc cgctggcgtg
     1201 cggtgcgacc accctgatgt tcgaaggtgt tccgaactgg ccgaccccgt ctcgtatggc
     1261 gcaggttgtt gacaaacacc aggttaacat cctgtatacc gcgccgaccg cgatccgtgc
     1321 gctgatggcg gaaggtgaca aagcgatcga aggtaccgac cgttcttctc tgcgtatcct
     1381 gggttctttt ggtgaaccga tcaacccgga agcgtgggaa tggtactgga aaaaaatcgg
     1441 taacgaacgt tgcccggttg ttgacacctg gtggcagacc gaaaccggtg gtgcgatgat
     1501 caccccgctg ccgggtgcga tcgaactgaa accgggttct gcgacccgtc cgttcttcgg
     1561 tgttcagccg gcgctggttg acaacgaagg taacccgcag gaaggtgcga ccgaaggtaa
     1621 cctggttatc accgactctt ggccgggtca ggcgcgtacc ctgtacggtg accacgaacg
     1681 tttcgaacag acctacttct ctaccttcaa aggtatgtac ttctctggtg acggtgcgcg
     1741 tcgtgacgaa gacggttact actggatcac cggtcgtgtt gacgacgttc tgaacgtttc
     1801 tggtcaccgt ctgggtaccg cggaaatcga atctgcgctg gttgcgcacc cgaaaatcgc
     1861 ggaagcggcg gttgttggta tcccgcacga catcaaaggt caggcgatct acgcgtacgt
     1921 taccctgaac cacggtgaag aaccgtctcc ggaactgtac aaagaagttc gtaactgggt
     1981 tcgtaaagaa atcggtccgc tggcgacccc ggacgttctg cactggaccg acggtctgcc
     2041 gaaaacccgt tctggtaaaa tcatgcgtcg tatcctgcgt aaaatcgcgg cgggtgacac
     2101 ctctaacctg ggtgacacct ctaccctggc ggacccgtct gttgttgaca aactgatcga
     2161 agaaaaacag gcgatcgcga tgccgtctct cgagcaccac caccaccacc actgagatcc
     2221 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     2281 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     2341 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2401 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2461 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2521 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2581 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2641 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     2701 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     2761 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     2821 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2881 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     2941 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     3001 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     3061 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     3121 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     3181 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     3241 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     3301 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3361 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     3421 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3481 ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     3541 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     3601 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3661 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     3721 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     3781 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     3841 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     3901 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3961 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4021 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4081 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     4141 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4201 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4261 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4321 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4381 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4441 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4501 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4561 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4621 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4681 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4741 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4801 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     4861 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     4921 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     4981 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     5041 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     5101 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     5161 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     5221 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     5281 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     5341 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     5401 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     5461 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     5521 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     5581 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     5641 atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     5701 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     5761 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     5821 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     5881 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     5941 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     6001 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     6061 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     6121 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     6181 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     6241 gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga
     6301 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     6361 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     6421 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     6481 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     6541 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     6601 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     6661 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     6721 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     6781 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     6841 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     6901 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     6961 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     7021 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     7081 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     7141 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
     7201 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
     7261 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaa
//
