LOCUS       sequence-311408-        6214 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses gSTEP1 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6214)
  AUTHORS   Eason MG, Pandelieva AT, Mayer MM, Khan ST, Garcia HG, Chica RA
  TITLE     Genetically Encoded Fluorescent Biosensor for Rapid Detection of 
            Protein Expression.
  JOURNAL   ACS Synth Biol. 2020 Nov 20;9(11):2955-2963. doi: 
            10.1021/acssynbio.0c00407. Epub 2020 Oct 12.
  PUBMED    33044070
REFERENCE   2  (bases 1 to 6214)
  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: 
            "10.1021/acssynbio.0c00407"; journalName: "ACS Synth Biol"; date: 
            "2020-11-20- 20"; volume: "9"; issue: "11"; pages: "2955-2963"
COMMENT     Sequence Label: pET-gSTEP1
FEATURES             Location/Qualifiers
     source          1..6214
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        214..232
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    233..257
                     /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             303..320
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1152..1196
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1234..1252)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1248..1295
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1332..1787
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1419..1438)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1629..1650
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1902..2693,2694..2762))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2525..2544
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2763..2855)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2936..3524
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3425..3444
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3689..3706
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3721..3860
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3786..3805)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3946..3968
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3962..4153)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4725..5807)
                     /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     5769..5788
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5808..5885)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6091..6110
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(6198..6214,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag
      121 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
      181 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
      241 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
      301 tgcatcacca ccatcaccat gacttgcgcc ctgagatttg gattgcacaa gagctgcgtc
      361 gtattggtga cgaattcaat gcgtattatg cgcgtcgtca caagatccgc ctggaaaatg
      421 tttacattaa agccgataaa cagaaaaacg gcatcaaagc aaacttcaag atccgccaca
      481 acatcgagga tggtggcgtt cagctggcct atcattacca gcagaacacc ccgattggtg
      541 acggtccggt cctgctgccg gacaaccact atttgtccgt gcagtcgaag ttgagcaaag
      601 atccgaatga aaaacgtgat cacatggtgc tgctggagtt tgttacggct gcgggtatta
      661 ccctgggtat ggatgaactg tacaagggtg gtactggtgg tagcatggtg agcaaaggcg
      721 aagaactgtt cacgggtgtc gttccgatcc tggttgagtt ggacggtgac gttaacggcc
      781 acaagtttag cgtgagcggc gagggtgagg gtgatgcgac ctatggcaag ctgaccctga
      841 aattcatttg caccacgggc aagctgccgg tcccgtggcc gaccctggtg accaccctga
      901 cgtacggtgt ccagtgcttt tctcgctacc cggaccacat gaagcaacat gacttcttta
      961 agtccgcgat gccagagggc tatatccaag agcgtactat cttctttaaa gacgatggta
     1021 actataagac ccgcgctgaa gtcaagttcg agggcgatac gttggtcaat cgtattgaac
     1081 tgaagggcat cgatttcaaa gaggacggta atatcctggg tcacaaactg gagtactgac
     1141 tcgagtctgg taaagaaacc gctgctgcga aatttgaacg ccagcacatg gactcgtcta
     1201 ctagcgcagc ttaattaacc taggctgctg ccaccgctga gcaataacta gcataacccc
     1261 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     1321 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     1381 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     1441 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     1501 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     1561 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     1621 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     1681 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     1741 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttctg gcggcacgat
     1801 ggcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     1861 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     1921 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     1981 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     2041 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     2101 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     2161 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     2221 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     2281 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     2341 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     2401 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     2461 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     2521 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     2581 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     2641 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     2701 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     2761 atactcttcc tttttcaatc atgattgaag catttatcag ggttattgtc tcatgagcgg
     2821 atacatattt gaatgtattt agaaaaataa acaaataggt catgaccaaa atcccttaac
     2881 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     2941 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3001 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3061 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     3121 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3181 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3241 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3301 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3361 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3421 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3481 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3541 cctttttacg gttcctggcc ttttgctggc cttttgctgg ccttttgctc acatgttctt
     3601 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     3661 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     3721 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     3781 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     3841 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     3901 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     3961 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     4021 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     4081 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     4141 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     4201 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     4261 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     4321 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     4381 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     4441 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     4501 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     4561 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg ctagtcatgc
     4621 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     4681 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     4741 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4801 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     4861 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     4921 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     4981 gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg
     5041 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     5101 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     5161 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     5221 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     5281 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     5341 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     5401 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     5461 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     5521 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     5581 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     5641 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     5701 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     5761 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     5821 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     5881 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     5941 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     6001 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     6061 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     6121 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga
     6181 tctcgatccc gcgaaattaa tacgactcac tata
//
