LOCUS       sequence-493947-        5983 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for growth-based enrichment of functional transcription factor 
            variants and fluorescent-based readout of transcription factor 
            function..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5983)
  TITLE     Example of directed evolution in E. coli to develop novel 
            ligand-binding regulators: evolving a progesterone-responsive 
            transcription factor to bind cortisol
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5983)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pSELIS-SRTF1
FEATURES             Location/Qualifiers
     source          1..5983
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      63..608
                     /direction=RIGHT
                     /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     509..528
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     725..742
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1134..1236
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1237..1896
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISRWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1303..1322)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        2734..2768
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     CDS             2819..3529
                     /codon_start=1
                     /gene="cI"
                     /product="phage lambda repressor"
                     /label=lambda repressor
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLAKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETF"
     promoter        3655..3689
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     RBS             3725..3733
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             4463..4480
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4495..4566
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4582..4609
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        4661..4738
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4739..5821
                     /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="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKAEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGLLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDHLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4758..4777)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tttgaagaga taaattgcac tgaaatctag aaatatttta tctgattaat aagatgatct
       61 tcttgagatc gttttggtct gcgcgtaatc tcttgctctg aaaacgaaaa aaccgccttg
      121 cagggcggtt tttcgaaggt tctctgagct accaactctt tgaaccgagg taactggctt
      181 ggaggagcgc agtcaccaaa acttgtcctt tcagtttagc cttaaccggc gcatgacttc
      241 aagactaact cctctaaatc aattaccagt ggctgctgcc agtggtgctt ttgcatgtct
      301 ttccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg actgaacggg
      361 gggttcgtgc atacagtcca gcttggagcg aactgcctac ccggaactga gtgttaggcg
      421 tggaatgaga caaacgcggc cataacagcg gaatgacacc ggtaaaccga aaggcaggaa
      481 caggagagcg cacgagggag ccaccagggg gaaacgcctg gtatctttat agtcctgtcg
      541 ggtttcgcca ccactgattt gagcgtcaga tttcgtgatg cttgtcaggg gggcggagcc
      601 tatggaaaaa cggctttgcc gcggccctct cacttccctg ttaagtatct tcctggcatc
      661 ttccaggaaa tctccgcccc gttcgtaagc catttccgct cgccgcagtc gaacgaccga
      721 gcgtagcgag tcagtgagcg aggaagcgga atatatcctg tatcacatat tctgctgacg
      781 caccggtgca gccttttttc tcctgccaca tgaagcactt cactgacacc ctcatcagtg
      841 ccaacatagt aagccagtat acactccgct agcgctgatg tccggcggtg cttttgccgt
      901 tacgcaccac cccgtcagta gctgaacagg agggacagct gatagaaaca gaagccactg
      961 gagcacctca aaaacaccat catacactaa atcagtaagt tggcagcatc acccgacgca
     1021 ctttgcgccg aataaatacc tgtgacggaa gatcacttcg cagaataaat aaatcctggt
     1081 gtccctgttg ataccgggaa gccctgggcc aacttttggc gaaaatgaga cgttgatcgg
     1141 cacgtaagag gttccaactt tcaccataat gaaataagat cactaccggg cgtatttttt
     1201 gagttatcga gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat
     1261 accaccgttg atatatcccg atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt
     1321 gctcaatgta cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta
     1381 aagaaaaata agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat
     1441 gctcatccgg agttccgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt
     1501 cacccttgtt acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa
     1561 taccacgacg atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt
     1621 gaaaacctgg cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat
     1681 ccctgggtga gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc
     1741 cccgttttca ctatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg
     1801 attcaggttc atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct taatgaatta
     1861 caacagtact gcgatgagtg gcagggcggg gcgtaatttt tttaaggcag ttattggtgc
     1921 ccttaaacgc ctggttgcta cgcctgaata agtgataata agcggatgaa tggcagaaat
     1981 tcgaaagcaa attcgacccg gtcgtcggtt cagggcaggg tcgttaaata gccgcttatg
     2041 tctattgctg gtttaccggt ttattgacta ccggaagcag tgtgaccgtg tgcttctcaa
     2101 atgcctgagg tttcagaaat aataaaaaag ccggattaat aatctggctt tttatattct
     2161 ctctttatta atcttgttct tcagcaacaa aatgaacgca attaccagct ggatctctca
     2221 aagcaaattc tctaccccaa ggttgttcac caatttcggt catagctgga ccagaagcat
     2281 ctctaaaatt ggtagaaaca acttcggacc attcggcata taattcatcc aaacctctaa
     2341 cccaaaccca agccaaagta ttatctggaa caacttgatc ttgaacggcg gaaatgaaca
     2401 aggtaacatc atctctaaca acaccagcga aatcatcttc aacgaaatct ctggagaaac
     2461 ccaatctatc agtccaaaat tcaacagcac cagcaacatc tctagcggtc aaaactggaa
     2521 cagcagatgt caatttagac atatgtcaac ctcctccaat aaccgtcact gctgacgact
     2581 ctaccattat cacttgcaac cattatcacc gccagaggta aaatagtcaa cacgcacggt
     2641 gttagtcttt tcgccggtgt atttcttgca aaaaaaaacc ccgccgaagc ggggtttttt
     2701 ttttacaggg ctataaggta gatccgggag ctgttgacag ctagctcagt cctaggtata
     2761 atgctagcta actagccgtt cggctagtta aatcaagtac tagagaagga gatctagaat
     2821 gagcacaaaa aagaaaccat taacacaaga gcagcttgag gacgcacgtc gccttaaagc
     2881 aatttatgaa aaaaagaaaa atgaacttgg cttatcccag gaatctgtcg cagacaagat
     2941 ggggatgggg cagtcaggcg ttggtgcttt atttaatggc atcaatgcat taaatgctta
     3001 taacgccgca ttgcttgcaa aaattctcaa agttagcgtt gaagaattta gcccttcaat
     3061 cgccagagaa atctacgaga tgtatgaagc ggttagtatg cagccgtcac ttagaagtga
     3121 gtatgagtac cctgtttttt ctcatgttca ggcagggatg ttctcacctg agcttagaac
     3181 ctttaccaaa ggtgatgcgg agagatgggt aagcacaacc aaaaaagcca gtgattctgc
     3241 attctggctt gaggttgaag gtaattccat gaccgcacca acaggctcca agccaagctt
     3301 tcctgacgga atgttaattc tcgttgaccc tgagcaggct gttgagccag gtgatttctg
     3361 catagccaga cttgggggtg atgagtttac cttcaagaaa ctgatcaggg atagcggtca
     3421 ggtgttttta caaccactaa acccacagta cccaatgatc ccatgcaatg agagttgttc
     3481 cgttgtgggg aaagttatcg ctagtcagtg gcctgaagag acgttttaac gataatgtcg
     3541 ggcaatcagg tgcgactcgg taccaaattc cagaaaagag gggagcggga aaccgctccc
     3601 cttttttcgt tttggtccca atctatcgat tgtatgggct gtcgaccttt gaaattgaca
     3661 gctagctcag tcctaggtat aatgctagct aactagccgt tcggctagtt agggtttgat
     3721 ttaataagga ggtatacata tggcatccaa gggcgaggag ctctttactg gcgtagtacc
     3781 aattctcgta gagctcgatg gcgatgtaaa tggccataag ttttccgtac gcggcgaggg
     3841 cgagggcgat gcaactaacg gcaagctcac tctcaagttt atttgtacta ctggcaagct
     3901 cccagtacca tggccaactc tcgtaactac tctgacctat ggcgtacaat gtttttcccg
     3961 ctatccagat cacatgaagc aacatgattt ttttaagtcc gcaatgccag agggctatgt
     4021 acaagagcgc actattagct ttaaggatga tggcacctat aagactcgcg cagaggtaaa
     4081 gtttgagggc gatactctcg taaatcgcat tgagctcaag ggcattgatt ttaaggagga
     4141 tggcaatatt ctcggccata agctggagta taatttcaat tcccataatg tatatattac
     4201 cgcagataag caaaagaatg gcattaaggc gaattttaag attcgccata atgtggagga
     4261 tggctccgta caactcgcag atcattatca acaaaatact ccaattggcg atggcccagt
     4321 actcctccca gataatcatt atctctccac tcaatccgtg ctctccaaag atccaaatga
     4381 gaagcgcgat cacatggtac tcctggagtt tgtaactgca gcaggcatta ctcatggcat
     4441 ggatgagctc tataagctcg agcaccacca ccaccaccac taataaccag gcatcaaata
     4501 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     4561 gctctctact agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttataatttc
     4621 ctaatgcagg agtcgcataa gggagagcgt cgagatcccg gacaccatcg aatggcgcaa
     4681 aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt
     4741 gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc
     4801 ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag cggaagcggc
     4861 gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc
     4921 gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc
     4981 ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg
     5041 aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg
     5101 gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac
     5161 taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt
     5221 ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca
     5281 aatcgcgctg ttagcgggcc tattaagttc tgtctcggcg cgtctgcgtc tggctggctg
     5341 gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag
     5401 tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc
     5461 gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg
     5521 gctgcgcgtt ggtgcggaca tctcggtagt gggatacgac gataccgaag acagctcatg
     5581 ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt
     5641 ggaccacttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt
     5701 ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc
     5761 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     5821 agcgcaacgc aattaatgta agttagctca ctcattaggc accgggatct cgaccgatgc
     5881 ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg actaacatga
     5941 gaattacaac ttatatcgta tggggctgac ttcaggtgct aca
//
