LOCUS       sequence-175689-        5222 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial co-expression of folA (DHFR) and thyA(TS) with a barcode 
            within the noncoding region between the genes .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5222)
  AUTHORS   Schober AF, Mathis AD, Ingle C, Park JO, Chen L, Rabinowitz JD, 
            Junier I, Rivoire O, Reynolds KA
  TITLE     A Two-Enzyme Adaptive Unit within Bacterial Folate Metabolism.
  JOURNAL   Cell Rep. 2019 Jun 11;27(11):3359-3370.e7. doi: 
            10.1016/j.celrep.2019.05.030.
  PUBMED    31189117
REFERENCE   2  (bases 1 to 5222)
  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.celrep.2019.05"; journalName: "Cell Rep"; date: 
            "2019-06-11- 11"; volume: "27"; issue: "11"; pages: "3359-3370.e7"
COMMENT     Sequence Label: pACYCDuet_F31VDHFR_WTTS
FEATURES             Location/Qualifiers
     source          1..5222
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        77..95
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    96..120
                     /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)."
     CDS             982..1026
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1064..1082)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1078..1125
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1345..2004)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1919..1938
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2005..2107)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2499..2516)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2633..3177)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(2713..2732)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3402..4484)
                     /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     4446..4465
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4485..4562)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        4608..4626
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4627..4651
                     /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)."
     CDS             4707..4724
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4737..5216
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDRVIGMENAMPWNLPADLAWVKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
ORIGIN
        1 tcgacaagct tgcggccgca taatgcttaa gtcgaacaga aagtaatcgt attgtacacg
       61 gccgcataat cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
      121 ccatcttagt atattagtta agtataagaa ggagatatac atatggcaga tctcatgaaa
      181 cagtacctgg agctgatgca aaaagttctg gatgagggta cccagaaaaa cgaccgcacg
      241 ggtaccggaa cgctgagcat ttttggccat cagatgcgtt ttaacctgca ggacggattc
      301 ccgctggtta cgaccaaacg ctgccacctg cgtagcatta ttcatgagct gctgtggttt
      361 ctgcaaggtg acactaacat cgcgtatctg cacgaaaaca atgtgacgat ctgggatgaa
      421 tgggccgatg aaaacggcga tctgggccca gtgtatggta aacagtggcg cgcctggcca
      481 acgccagatg gccgccacat cgatcagatc acgaccgtgc tgaatcaact gaaaaacgac
      541 ccggacagcc gccgcattat tgtttccgcg tggaatgtgg gtgaactgga taaaatggcg
      601 ctggcgccgt gccatgcatt cttccagttt tacgttgcgg acggtaagct gagctgtcaa
      661 ctttatcagc gcagctgcga tgttttcctc ggcctgccgt tcaatatcgc cagctacgcg
      721 ttactggtgc atatgatggc gcagcagtgc gacctggagg tgggtgattt tgtctggacc
      781 ggcggtgata cccacctgta cagcaaccat atggaccaaa cgcatctgca attgagccgt
      841 gagccgcgtc cgctgccgaa actgattatt aagcgcaaac ccgagagcat ctttgactat
      901 cgtttcgaag acttcgagat tgaaggctac gatccgcacc cgggtatcaa agcgccggtg
      961 gctatctaac tcgagtctgg taaagaaacc gctgctgcga aatttgaacg ccagcacatg
     1021 gactcgtcta ctagcgcagc ttaattaacc taggctgctg ccaccgctga gcaataacta
     1081 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa acctcaggca
     1141 tttgagaagc acacggtcac actgcttccg gtagtcaata aaccggtaaa ccagcaatag
     1201 acataagcgg ctatttaacg accctgccct gaaccgacga ccgggtcgaa tttgctttcg
     1261 aatttctgcc attcatccgc ttattatcac ttattcaggc gtagcaccag gcgtttaagg
     1321 gcaccaataa ctgccttaaa aaaattacgc cccgccctgc cactcatcgc agtactgttg
     1381 taattcatta agcattctgc cgacatggaa gccatcacag acggcatgat gaacctgaat
     1441 cgccagcggc atcagcacct tgtcgccttg cgtataatat ttgcccatag tgaaaacggg
     1501 ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg
     1561 attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc
     1621 accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta
     1681 ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg
     1741 aacactatcc catatcacca gctcaccgtc tttcattgcc atacggaact ccggatgagc
     1801 attcatcagg cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt
     1861 tacggtcttt aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc
     1921 aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt
     1981 atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc tcgataactc
     2041 aaaaaatacg cccggtagtg atcttatttc attatggtga aagttggaac ctcttacgtg
     2101 ccgatcaacg tctcattttc gccaaaagtt ggcccagggc ttcccggtat caacagggac
     2161 accaggattt atttattctg cgaagtgatc ttccgtcaca ggtatttatt cggcgcaaag
     2221 tgcgtcgggt gatgctgcca acttactgat ttagtgtatg atggtgtttt tgaggtgctc
     2281 cagtggcttc tgtttctatc agctgtccct cctgttcagc tactgacggg gtggtgcgta
     2341 acggcaaaag caccgccgga catcagcgct agcggagtgt atactggctt actatgttgg
     2401 cactgatgag ggtgtcagtg aagtgcttca tgtggcagga gaaaaaaggc tgcaccggtg
     2461 cgtcagcaga atatgtgata caggatatat tccgcttcct cgctcactga ctcgctacgc
     2521 tcggtcgttc gactgcggcg agcggaaatg gcttacgaac ggggcggaga tttcctggaa
     2581 gatgccagga agatacttaa cagggaagtg agagggccgc ggcaaagccg tttttccata
     2641 ggctccgccc ccctgacaag catcacgaaa tctgacgctc aaatcagtgg tggcgaaacc
     2701 cgacaggact ataaagatac caggcgtttc ccctggcggc tccctcgtgc gctctcctgt
     2761 tcctgccttt cggtttaccg gtgtcattcc gctgttatgg ccgcgtttgt ctcattccac
     2821 gcctgacact cagttccggg taggcagttc gctccaagct ggactgtatg cacgaacccc
     2881 ccgttcagtc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggaaa
     2941 gacatgcaaa agcaccactg gcagcagcca ctggtaattg atttagagga gttagtcttg
     3001 aagtcatgcg ccggttaagg ctaaactgaa aggacaagtt ttggtgactg cgctcctcca
     3061 agccagttac ctcggttcaa agagttggta gctcagagaa ccttcgaaaa accgccctgc
     3121 aaggcggttt tttcgttttc agagcaagag attacgcgca gaccaaaacg atctcaagaa
     3181 gatcatctta ttaatcagat aaaatatttc tagatttcag tgcaatttat ctcttcaaat
     3241 gtagcacctg aagtcagccc catacgatat aagttgtaat tctcatgtta gtcatgcccc
     3301 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     3361 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     3421 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     3481 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     3541 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     3601 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     3661 tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg
     3721 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     3781 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     3841 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     3901 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     3961 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     4021 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     4081 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     4141 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     4201 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     4261 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     4321 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     4381 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     4441 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     4501 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     4561 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaaattaa tacgactcac
     4621 tataggggaa ttgtgagcgg ataacaattc ccctgtagaa ataattttgt ttaactttaa
     4681 taaggagata taccatgggc agcagccatc accatcatca ccacagccag gatccgatga
     4741 tcagtctgat tgcggcgtta gcggtagatc gcgttatcgg catggaaaac gccatgccgt
     4801 ggaacctgcc tgccgatctc gcctgggtga aacgcaacac cttaaataaa cccgtgatta
     4861 tgggccgcca tacctgggaa tcaatcggtc gtccgttgcc aggacgcaaa aatattatcc
     4921 tcagcagtca accgggtacg gacgatcgcg taacgtgggt gaagtcggtg gatgaagcca
     4981 tcgcggcgtg tggtgacgta ccagaaatca tggtgattgg cggcggtcgc gtttatgaac
     5041 agttcttgcc aaaagcgcaa aaactgtatc tgacgcatat cgacgcagaa gtggaaggcg
     5101 acacccattt cccggattac gagccggatg actgggaatc ggtattcagc gaattccacg
     5161 atgctgatgc gcagaactct cacagctatt gctttgagat tctggagcgg cggtaaccga
     5221 ag
//
