LOCUS       sequence-90573-e        4646 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP from jellyfish Aquorea sp. under T5 promoter with lac operators;
            kanamycin resistant..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4646)
  AUTHORS   Siddall A, Williams AA, Sanders J, Denton JA, Madden D, Schollar J, 
            Bryk J
  TITLE     UNIGEMS: plasmids and parts to facilitate teaching on assembly, gene
            expression control and logic in E. coli
  JOURNAL   bioRxiv 2021.06.20.449138
REFERENCE   2  (bases 1 to 4646)
  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: "bioRxiv 
            2021.06.20.449138"
COMMENT     Sequence Label: p006kanGFP
FEATURES             Location/Qualifiers
     source          1..4646
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      476..1064
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     965..984
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1218..1235
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1333..1350
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1334..1350
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1807..1823
                     /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)."
     promoter        join(1848..1862,1863..1868,1869..1885,1886..1892)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    1868..1884
                     /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)."
     protein_bind    1898..1917
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     RBS             1958..1966
                     /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             1972..2688
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTRVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKVRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     2625..2645
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(2884..2903)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        2970..3061
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3062..3871
                     /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="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             join(3954..4646,1..390)
                     /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     complement(3973..3992)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
       61 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg
      121 ggatacgacg ataccgaaga tagctcatgt tatatcccgc cgttaaccac catcaaacag
      181 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
      241 gcggtgaagg gcaatcagct gttgccagtc tcactggtga aaagaaaaac caccctggcg
      301 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
      361 caggtttccc gactggaaag cgggcagtga ctcatgacca aaatccctta acgtgagtta
      421 cgcgcgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
      481 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
      541 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
      601 gagcgcagat accaaatact gttcttctag tgtagccgta gttagcccac cacttcaaga
      661 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
      721 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
      781 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
      841 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
      901 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
      961 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     1021 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     1081 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     1141 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     1201 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaaggcgag agtagggaac
     1261 tgccaggcat caaactaagc agaaggcccc tgacggatgg cctttttgcg tttctacaaa
     1321 ctctttctgt gttgtaaaac gacggccagt cttaagctcg ggccccctgg gcggttctga
     1381 taacgagtaa tcgttaatcc gcaaataacg taaaaacccg cttcggcggg tttttttatg
     1441 gggggagttt agggaaagag catttgtcag aatatttaag ggcgcctgtc actttgcttg
     1501 atatatgaga attatttaac cttataaatg agaaaaaagc aacgcacttt aaataagata
     1561 cgttgctttt tcgattgatg aacacctata attaaactat tcatctatta tttatgattt
     1621 tttgtatata caatatttct agtttgttaa agagaattaa gaaaataaat ctcgaaaata
     1681 ataaagggaa aatcagtttt tgatatcaaa attatacatg tcaacgataa tacaaaatat
     1741 aatacaaact ataagatgtt atcagtattt attatgcatt tagaataaat tttgtgtcgc
     1801 ccttaattgt gagcggataa caattacgag cttcatgcac agtgaaatca tgaaaaattt
     1861 atttgctttg tgagcggata acaattataa tatgtggaat tgtgagcgct cacaattcca
     1921 caacggtttc cctctagaaa taattttgtt taacttttaa ggaggtaaaa aatgagtaaa
     1981 ggagaagaac ttttcactag agttgtccca attcttgttg aattagacgg tgatgttaat
     2041 gggcacaaat tttctgtcag tggagaggga gaaggtgatg caacatacgg aaaacttacc
     2101 cttaaattta tttgcactac tggaaaacta ccagttccgt ggccaacact tgtcactact
     2161 ctctcttatg gtgttcaatg cttttcgaga tacccagatc acatgaaacg gcatgacttt
     2221 ttcaagagtg ccatgcccga gggttatgta caggaaagaa ctatattttt caaagatgac
     2281 gggaactaca agacacgtgc tgaagtcaag tttgaaggtg atacccttgt taatagaatc
     2341 gagttaaaag gtgttgattt taaagaagat ggaaacattc ttggacacaa attggaatac
     2401 aactataact cacacaatgt atacatcatg gcagacaaac aaaagaatgg aatcaaagct
     2461 aacttcaaag ttagacacaa cattgaagat ggaagcgttc aactggcaga ccattatcaa
     2521 caaaatactc caattggcga tggccctgtc cttttaccag acaaccatta cctgtccaca
     2581 caatctgccc tttcgaaaga tcccaacgaa aagagagacc acatggtcct tcttgagttt
     2641 gtaacagctg ctgggattac acatggcatg gatgaactat acaaataacc ccaagggcga
     2701 caccccctaa ttagcccggg cgaaaggccc agtctttcga ctgagccttt cgttttattt
     2761 gatgcctggc agttccctac tctcgcatgg ggagtcccca cactaccatc ggcgctacgg
     2821 cgtttcactt ctgagttcgg catggggtca ggtgggacca ccgcgctact gccgccaggc
     2881 aaacaagggg tgttatgagc catattcagg tataaatggg ctcgcgataa tgttcagaat
     2941 tggttaattg gttgtaacac tgacccctat ttgtttattt ttctaaatac attcaaatat
     3001 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagaa
     3061 tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga
     3121 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
     3181 cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
     3241 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgccacttcc
     3301 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc
     3361 cggaaaaaca gcgttccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
     3421 tgcgctggca gtgttcctgc gccggttgca ctcgattcct gtttgtaatt gtccttttaa
     3481 cagcgatcgc gtatttcgcc tcgctcaggc gcaatcacga atgaataacg gtttggttga
     3541 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
     3601 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
     3661 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
     3721 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
     3781 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
     3841 gtttcatttg atgctcgatg agtttttcta agcggcgcgc catcgaatgg cgcaaaacct
     3901 ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatatgaaac
     3961 cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg
     4021 tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg
     4081 cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc
     4141 tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga
     4201 ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg
     4261 gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga
     4321 tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg
     4381 ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt attttctccc
     4441 atgaggacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg
     4501 cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata
     4561 aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca
     4621 tgtccggttt tcaacaaacc atgcaa
//
