LOCUS       sequence-213525-        7319 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Funclib design ACS_9.
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_9
FEATURES             Location/Qualifiers
     source          1..7319
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     12..31
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     115..134
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        115..133
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    134..158
                     /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             173..195
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2165..2182
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2235..2253)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2249..2296
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2333..2788
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2420..2439)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2630..2651
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2814..2918
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2919..2987,2988..3779)
                     /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(3137..3156)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3950..4538
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4439..4458
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4692..4709
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4724..4863
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4789..4808)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4949..4971
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4965..5156)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5965..7047)
                     /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     7009..7028
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7048..7125)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
       61 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac
      121 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
      181 actttaagaa ggagatatac atatgtctca gacccacaaa cacccggttc cgccgaacat
      241 cgcggcgaac gcgtggatca acccggaaca gtacgaagcg atgtacaaac agtctatcaa
      301 cgacccggac accttctggc gtgaacaggg taaaatcctg gactggatca aaccgtacac
      361 ccgtgttaaa aacacctctt tcgaaccggg taacgtttct atccgttggt acgaagacgg
      421 taccctgaac ctggcgtaca actgcctgga ccgtcacctg gaagaacgtg gtgaccagac
      481 cgcgatcatc tgggaaggtg acgacccgtc tgaatctcgt cacatcacct accgtgaact
      541 gcaccgtgac gtttgccgtt tcgcgaacgt tctgaaagcg ctgggtatca aaaaaggtga
      601 cgttgttgcg atctacatgc cgatggttcc ggaagcggcg gttgcgatgc tggcgtgcgc
      661 gcgtatcggt gcgatccact ctgttatctt cggtggtttc tctccggaag cggttgcggg
      721 tcgtatcatc gactctaact ctcgtctggt tatcaccgcg gacgaaggtg ttcgtgcggg
      781 tcgttctatc ccgctgaaaa agaacgttga cgaagcgctg aaaaacccga acgttacctc
      841 tgttgaacac gttatcgttt tcaaacgtac cggtggtgac atcgactggc aggaaggtcg
      901 tgacctgtgg tggcgtgacc tgatcgaaaa agcgtctccg gaacaccagc cggaaccgat
      961 gaacgcggaa gacccgctgt tcatcctgta cacctctggt tctaccggta aaccgaaagg
     1021 tgttctgcac accaccggtg gttacctggt ttacgcggcg accaccttca aatacgtttt
     1081 cgactaccac ccgggtgaca tctactggtg caccgcggac gttggttggg cggcgggtca
     1141 cacctatctg ctgtacggtc cgctggcgtg cggtgcgacc accctgatgt tcgaaggtgt
     1201 tccgaactgg ccgaccccgt ctcgtatggc gcaggttgtt gacaaacacc aggttaacat
     1261 cctgtttacc gcgccgaccg cgatccgtgc gctgatggcg gaaggtgaca aagcgatcga
     1321 aggtaccgac cgttcttctc tgcgtatcct gggttctctg ggtgaaccga tcaacccgga
     1381 agcgtgggaa tggtactgga aaaaaatcgg taacgaacgt tgcccggttg ttgacacctg
     1441 gtggcagacc gaaaccggtg gtgcgatgat caccccgctg ccgggtgcga tcgaactgaa
     1501 accgggttct gcgacccgtc cgttcttcgg tgttcagccg gcgctggttg acaacgaagg
     1561 taacccgcag gaaggtgcga ccgaaggtaa cctggttatc accgactctt ggccgggtca
     1621 ggcgcgtacc ctgtacggtg accacgaacg tttcgaacag acctacttct ctaccttcaa
     1681 aggtatgtac ttctctggtg acggtgcgcg tcgtgacgaa gacggttact actggatcac
     1741 cggtcgtgtt gacgacgttc tgaacgtttc tggtcaccgt ctgggtaccg cggaaatcga
     1801 atctgcgctg gttgcgcacc cgaaaatcgc ggaagcggcg gttgttggta tcccgcacga
     1861 catcaaaggt caggcgatct acgcgtacgt taccctgaac cacggtgaag aaccgtctcc
     1921 ggaactgtac aaagaagttc gtaactgggt tcgtaaagaa atcggtccgc tggcgacccc
     1981 ggacgttctg cactggaccg acggtctgcc gaaaacccgt tctggtaaaa tcatgcgtcg
     2041 tatcctgcgt aaaatcgcgg cgggtgacac ctctaacctg ggtgacacct ctaccctggc
     2101 ggacccgtct gttgttgaca aactgatcga agaaaaacag gcgatcgcga tgccgtctct
     2161 cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga
     2221 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     2281 cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc
     2341 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     2401 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     2461 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     2521 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     2581 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     2641 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     2701 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     2761 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     2821 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     2881 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     2941 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     3001 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     3061 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     3121 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     3181 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     3241 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     3301 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     3361 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     3421 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt
     3481 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     3541 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     3601 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     3661 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     3721 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     3781 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     3841 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     3901 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     3961 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4021 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4081 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4141 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     4201 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     4261 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     4321 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     4381 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     4441 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     4501 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     4561 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     4621 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     4681 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     4741 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     4801 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     4861 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     4921 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     4981 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     5041 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     5101 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     5161 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     5221 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     5281 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     5341 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     5401 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     5461 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     5521 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     5581 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     5641 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     5701 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     5761 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     5821 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     5881 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     5941 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     6001 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     6061 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     6121 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     6181 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     6241 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     6301 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     6361 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     6421 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     6481 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     6541 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     6601 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     6661 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     6721 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     6781 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     6841 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     6901 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     6961 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     7021 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     7081 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     7141 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     7201 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     7261 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccga
//
