LOCUS       sequence-57550-e        4511 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4511)
  AUTHORS   Moroz OV, Moroz YS, Wu Y, Olsen AB, Cheng H, Mack KL, McLaughlin JM,
            Raymond EA, Zhezherya K, Roder H, Korendovych IV
  TITLE     A Single Mutation in a Regulatory Protein Produces Evolvable 
            Allosterically Regulated Catalyst of Nonnatural Reaction.
  JOURNAL   Angew Chem Int Ed Engl. 2013 Apr 29. doi: 10.1002/anie.201302339.
  PUBMED    23630096
REFERENCE   2  (bases 1 to 4511)
  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"; journalName: "Angew Chem 
            Int Ed Engl. 2013 Apr 29. doi: 10.1002/anie.201302339."
COMMENT     Sequence Label: pMCSG49-AlleyCat7
FEATURES             Location/Qualifiers
     source          1..4511
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        285..388
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(389..457,458..1249)
                     /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(607..626)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1420..2008
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1909..1928
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2162..2179
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2262..2281)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(2430..3512)
                     /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     3474..3493
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3513..3590)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3796..3815
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3903..3922
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3903..3921
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3922..3946
                     /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             3961..3983
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3994..4011
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4036..4056
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             4355..4372
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4425..4443)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4439..4486
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
      301 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
      361 tgcttcaata atttgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      421 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      481 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      541 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      601 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
      661 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
      721 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      781 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
      841 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
      901 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
      961 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     1021 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     1081 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     1141 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1201 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1261 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1321 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1381 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1441 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1501 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1561 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1621 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1681 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1741 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     1801 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     1861 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     1921 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     1981 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     2041 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     2101 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     2161 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     2221 tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc
     2281 gcatagttaa gccagtatac actccgctat cgctactagt catgccccgc gcccaccgga
     2341 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     2401 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     2461 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     2521 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     2581 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     2641 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     2701 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     2761 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     2821 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     2881 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     2941 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     3001 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     3061 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     3121 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     3181 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     3241 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     3301 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     3361 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     3421 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     3481 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     3541 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     3601 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     3661 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     3721 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     3781 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     3841 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atcgatctcg atcccgcgaa
     3901 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat
     3961 tttgtttaac tttaagaagg agatatacat atgcaccatc atcatcatca ttcttctggt
     4021 gtagatctgg gtaccgagaa cctgtacttc caatccatga aagatacaga tagcgaagaa
     4081 gaacttagag aacagttccg tgtggaagac aaggatggta atggttacat tagtgctgca
     4141 gaacttcgta ttgtgatgac aaatcgtggg gagaagctaa cagatgaaga agttgatgaa
     4201 ctgattaggg aaacagacat tgatggtgat ggtcaagtaa actatgaaga gtttgtacaa
     4261 cgtatgacag cgaagtgaaa gggtgatccg gcattggaag tggataacgg atccgaattc
     4321 gagctccgtc gacaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc
     4381 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     4441 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     4501 tatatccgga t
//
