LOCUS       sequence-493103-        5522 bp    DNA     circular SYN 18-NOV-2025
DEFINITION  Final circuit with -Ara: OFF, +Ara: OFF behaviour.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1964 to 1998)
  AUTHORS   Nevot G, Guell M, Santos-Moreno J
  TITLE     Critical Analysis of Preprints and Inquiry-Based Lessons Improve the
            Synthetic Biology Learning Experience.
  JOURNAL   ACS Synth Biol. 2025 Aug 15;14(8):2878-2884. doi: 
            10.1021/acssynbio.5c00014.
  PUBMED    40814254
REFERENCE   2  (bases 1 to 5522)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 18, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1021/acssynbio";
            journalName: "ACS Synth Biol"; date: "2025-08-15- 15"; volume: "14";
            issue: "8"; pages: "2878-2884"
COMMENT     Sequence Label: pEND-11Y
FEATURES             Location/Qualifiers
     source          1..5522
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(364..1242)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1269..1553
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1476..1495
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     promoter        1964..1998
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     protein_bind    2142..2161
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             2166..2188
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2196..2909
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     promoter        3317..3351
                     /label=BBa_J23105
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.24"
     promoter        3317..3351
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     promoter        3317..3351
                     /label=BBa_J23101
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.7"
     protein_bind    3495..3514
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    3621..3640
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     terminator      3960..3987
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(4175..4990)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4175..4990)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4898..4917
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4991..5082)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(5100..5522,1..213))
                     /direction=LEFT
                     /label=ColA ori
                     /note="Plasmids containing the ColA origin of replication 
                     can be propagated in E. coli cells that contain additional 
                     plasmids with compatible origins."
     primer_bind     complement(5245..5264)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agtcttgaag tggtggccta acagaggcta cactgaaagg acagtatttg gtatctgcgc
       61 tccactaaag ccagttacca ggttaagcag ttccccaact gacttaacct tcgatcaaac
      121 cgcctcccca ggcggttttt tcgtttacag agcaggagat tacgacgatc gtaaaaggat
      181 ctcaagaaga tcctttacgg attcccgaca ccatcactct agatttcagt gcaatttatc
      241 tcttcaaatg tagcacctga agtcagcccc atacgatata agttgtaatt ctcatgttag
      301 tcatgccccg cgcccaccgg aaggagctga ctgggttgct cctagggtct gattcgttac
      361 caattatgac aacttgacgg ctacatcatt cactttttct tcacaaccgg cacggaactc
      421 gctcgggctg gccccggtgc attttttaaa tacccgcgag aaatagagtt gatcgtcaaa
      481 accaacattg cgaccgacgg tggcgatagg catccgggtg gtgctcaaaa gcagcttcgc
      541 ctggctgata cgttggtcct cgcgccagct taagacgcta atccctaact gctggcggaa
      601 aagatgtgac agacgcgacg gcgacaagca aacatgctgt gcgacgctgg cgatatcaaa
      661 attgctgtct gccaggtgat cgctgatgta ctgacaagcc tcgcgtaccc gattatccat
      721 cggtggatgg agcgactcgt taatcgcttc catgcgccgc agtaacaatt gctcaagcag
      781 atttatcgcc agcagctccg aatagcgccc ttccccttgc ccggcgttaa tgatttgccc
      841 aaacaggtcg ctgaaatgcg gctggtgcgc ttcatccggg cgaaagaacc ccgtattggc
      901 aaatattgac ggccagttaa gccattcatg ccagtaggcg cgcggacgaa agtaaaccca
      961 ctggtgatac cattcgcgag cctccggatg acgaccgtag tgatgaatct ctcctggcgg
     1021 gaacagcaaa atatcacccg gtcggcaaac aaattctcgt ccctgatttt tcaccacccc
     1081 ctgaccgcga atggtgagat tgagaatata acctttcatt cccagcggtc ggtcgataaa
     1141 aaaatcgaga taaccgttgg cctcaatcgg cgttaaaccc gccaccagat gggcattaaa
     1201 cgagtatccc ggcagcaggg gatcattttg cgcttcagcc atacttttca tactcccgcc
     1261 attcagagaa gaaaccaatt gtccatattg catcagacat tgccgtcact gcgtctttta
     1321 ctggctcttc tcgctaacca aaccggtaac cccgcttatt aaaagcattc tgtaacaaag
     1381 cgggaccaaa gccatgacaa aaacgcgtaa caaaagtgtc tataatcacg gcagaaaagt
     1441 ccacattgat tatttgcacg gcgtcacact ttgctatgcc atagcatttt tatccataag
     1501 attagcggtt cctacctgac gctttttatc gcaactctct actgtttctc cataccgaat
     1561 tcataggata gattctggaa actttaccgt ccgagctcag gcttacctta ctcgagcaat
     1621 aaacagttga tagggcttct ccgttaccat ggttcagcca aaaaacttaa gaccgccggt
     1681 cttgtccact accttgcagt aatgcggtgg acaggatcgg cggttttctt ttctcttctc
     1741 aattcttctg acctgtaacg aataatagat agtaaagtag tctccgattg agttttctct
     1801 gccgagtccc acccagttct gtgatttcag taagttggta attgatacac tgttgcgaga
     1861 actggtgcct ggtagtagat aggttgttat tgagtaagaa ggtaaagtga acgaaatccc
     1921 tgaaactgag actgtagaaa ataagcttca gcctgcggtc cggttgacgg ctagctcagt
     1981 cctaggtaca gtgctagctc gctgggacgc ccgagatagc cgttacacag gtgacactta
     2041 tttcagcctg cggtccggcc acactagagt ctagcttgag atcgctggga cgcccgtaga
     2101 actctgtgag gataaagtct cccttacagc ctgcggtccg ggttcactgc cgtataggca
     2161 gtaattttgt ttaactttaa gaaggagata tacatatgcg taaaggcgaa gaactgttta
     2221 ccggtgtggt tccgattctg gtggaactgg acggcgatgt taatggtcat aaattcagtg
     2281 ttcgcggcga aggtgaaggc gatgcgacga acggcaaact gaccctgaaa tttatctgca
     2341 ccacgggtaa actgccggtc ccgtggccga cgctggtgac cacgctgacc tatggcgttc
     2401 aatgttttgc gcgttacccg gatcacatga aacagcacga ctttttcaaa tcggccatgc
     2461 cggaaggcta tgtgcaggaa cgtacgatta gctttaaaga cgatggtacg tataaaaccc
     2521 gcgcggaagt gaaattcgaa ggcgataccc tggttaaccg tatcgaactg aaaggtatcg
     2581 atttcaaaga agacggcaat attctgggtc ataaactgga atataacttc aattcccaca
     2641 acgtgtacat caccgcggat aaacagaaaa acggcattaa agccaatttc aaaatccgcc
     2701 ataatgtgga agatggtagc gttcagctgg ccgaccacta tcagcaaaac acgccgattg
     2761 gtgatggccc ggtcctgctg ccggacaatc actacctgag tacccagtcc gtgctgtcaa
     2821 aagatccgaa cgaaaaacgt gaccacatgg tcctgctgga atttgtgacg gctgcgggta
     2881 tcacccacgg catggacgaa ctgtataaaa tgtcccgccg taatactgac gccatcacaa
     2941 tccacagcat cctggattgg attgaagact aataatcgct gggacgcccg ggatccaaga
     3001 gatttctaca cgattgagca ctgtctcctg caggctcggt accaaattcc agaaaagagg
     3061 cctcccgaaa ggggggcctt ttttcgtttt ggtcctaata gataaaggat aggtctggta
     3121 gtgttgttcg ttctcgcagg taaatcaata atactctgca gttccgtaga cttttcagtg
     3181 ggacagggta gcgataacag atagattgta ataagacaca gtaggtgctc gtagttgcgt
     3241 gaagagaacc gctcaggaaa tccagtcaga agtattggta atcgttgaaa actcagtcga
     3301 ccagcctgcg gtccggttta cggctagctc agtcctaggt attatgctag ctcgctggga
     3361 cgcccgtgaa cagttgctct gattgaaacc acgattcagc ctgcggtccg gccaagtact
     3421 tagtacacac tgattcgctg ggacgcccgg tagtgcttat cagacccaat actgttgaac
     3481 agcctgcggt ccgggttcac tgccgtatag gcagtctcaa gctagactct agtggtttca
     3541 gagctatgct ggaaacagca tagcaagttg aaataaggct agtccgttat caacttgaaa
     3601 aagtggcacc gagtcggtgc gttcactgcc gtataggcag tcgctgggac gcccggacgt
     3661 cctattacac tcgtcgttgg aaactgaaga tgcggccgcg gaaacacaga aaaaagcccg
     3721 cacctgacag tgcgggcttt ttttttcgac caaaggtagc gaacgacgag tcactgttga
     3781 ggataaatac tttctctact aggcgcctgt tacacaggtc ctcagcggcg cgcctttgtc
     3841 ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgtgaagca
     3901 acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc aaactaagca
     3961 gaaggccatc ctgacggatg gcctttttgc gtttcagatc taccggtaaa ccagcaatag
     4021 acataagcgg ctatttaacg accctgccct gaaccgacga caagctgacg accgggtctc
     4081 cgcaagtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     4141 acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat
     4201 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     4261 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     4321 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     4381 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     4441 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     4501 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcggt
     4561 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     4621 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     4681 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     4741 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     4801 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     4861 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     4921 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     4981 tatggctcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5041 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggcatg ctagcgcaga
     5101 aacgtcctag aagatgccag gaggatactt agcagagaga caataaggcc ggagcgaagc
     5161 cgtttttcca taggctccgc ccccctgacg aacatcacga aatctgacgc tcaaatcagt
     5221 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgat ggctccctct
     5281 tgcgctctcc tgttcccgtc ctgcggcgtc cgtgttgtgg tggaggcttt acccaaatca
     5341 ccacgtcccg ttccgtgtag acagttcgct ccaagctggg ctgtgtgcaa gaaccccccg
     5401 ttcagcccga ctgctgcgcc ttatccggta actatcatct tgagtccaac ccggaaagac
     5461 acgacaaaac gccactggca gcagccattg gtaactgaga attagtggat ttagatatcg
     5521 ag
//
