LOCUS       sequence-333378-        6203 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6203)
  AUTHORS   Cormack BP, Valdivia RH, Falkow S
  TITLE     FACS-optimized mutants of the green fluorescent protein (GFP).
  JOURNAL   Gene. 1996 . 173(1 Spec No):33-8.
  PUBMED    8707053
REFERENCE   2  (bases 1 to 6203)
  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: "Gene. 1996 
            . 173(1 Spec No):33-8."
COMMENT     Sequence Label: pKEN GFP mut2
FEATURES             Location/Qualifiers
     source          1..6203
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    355..371
                     /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        complement(383..401)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(408..424)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      565..1020
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(652..671)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     862..883
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1087..1106)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1206..1228
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1266..1284)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1352..1456
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1457..1525,1526..2317)
                     /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     complement(1675..1694)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2488..3076
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2977..2996
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3230..3247
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3405..3432)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3524..3610)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3763..3780
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(3844..4557)
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(3884..3904)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     protein_bind    5960..5976
                     /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        complement(5988..6006)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6013..6029)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gttctttcct gtacataacc ttcgggcatg gcactcttga aaaagtcatg ctgtttcata
       61 tgatctgggt atctcgcaaa gcattgaaga ccatacgcga aagtagtgac aagtgttggc
      121 catggaacag gtagttttcc agtagtgcaa ataaatttaa gggtaagttt tccgtatgtt
      181 gcatcacctt caccctctcc actgacagaa aatttgtgcc cattaacatc accatctaat
      241 tcaacaagaa ttgggacaac tccagtgaaa agttcttctc ctttactcat atgtatatct
      301 ccttcttaaa tctagaggat ccccgggtac cgagctcgaa ttccacacat tatattgtta
      361 tccgctcaca atgtcaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt
      421 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
      481 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
      541 ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
      601 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
      661 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
      721 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
      781 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
      841 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
      901 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
      961 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     1021 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac
     1081 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     1141 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     1201 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     1261 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     1321 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1381 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     1441 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     1501 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     1561 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     1621 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     1681 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     1741 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     1801 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     1861 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     1921 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     1981 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     2041 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     2101 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     2161 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     2221 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     2281 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     2341 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     2401 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     2461 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     2521 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2581 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     2641 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     2701 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     2761 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     2821 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     2881 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     2941 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3001 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3061 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3121 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3181 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3241 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     3301 tcattaatgc agttattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     3361 tgaatgtatt tagaaaaata aacaaaagag tttgtagaaa cgcaaaaagg ccatccgtca
     3421 ggatggcctt ctgcttaatt tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc
     3481 ctccgggccg ttgcttcgca acgttcaaat ccgctcccgg cggatttgtc ctactcagga
     3541 gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtctttcgac tgagcctttc
     3601 gttttatttg atgcctggca gttccctact ctcgcatggg gagaccccac actaccatcg
     3661 gcgctacggc gtttcacttc tgagttcggc atggggtcag gtgggaccac cgcgctactg
     3721 ccgccaggca aattctgttt tatcagaccg cttctgcgtt ctgatttaat ctgtatcagg
     3781 ctgaaaatct tctctcatcc gccaaaacag ccaagcttgc atgcctgcag gtctggacat
     3841 ttatttgtat agttcatcca tgccatgtgt aatcccagca gctgttacaa actcaagaag
     3901 gaccatgtgg tctctctttt cgttgggatc tttcgaaagg gcagattgtg tggacaggta
     3961 atggttgtct ggtaaaagga cagggccatc gccaattgga gtattttgtt gataatggtc
     4021 tgctagttga acgcttccat cttcaatgtt gtgtctaatt ttgaagttaa ctttgattcc
     4081 attcttttgt ttgtctgcca tgatgtatac attgtgtgag ttatagttgt attccaattt
     4141 gtgtccaaga atgtttccat cttctttaaa atcaatacct tttaactcga ttctattaac
     4201 aagggtatca ccttcaaact tgacttcagc acgtgtcttg tagttcccgt catctttgaa
     4261 aaatatagtt ctttcctgta cataaccttc gggcatggca ctcttgaaaa agtcatgctg
     4321 tttcatatga tctgggtatc tcgcaaagca ttgaagacca tacgcgaaag tagtgacaag
     4381 tgttggccat ggaacaggta gttttccagt agtgcaaata aatttaaggg taagttttcc
     4441 gtatgttgca tcaccttcac cctctccact gacagaaaat ttgtgcccat taacatcacc
     4501 atctaattca acaagaattg ggacaactcc agtgaaaagt tcttctcctt tactcatatg
     4561 tatatctcct tcttaaatct agaggatccc cgggtaccga gctctgagag atcccctcat
     4621 aatttcccca aagcgtaacc atgtgtgaat aaattttgag ctagtagggt tgcagccacg
     4681 agtaagtctt cccttgttat tgtgtagcca gaatgccgca aaacttccat gcctaagcga
     4741 actgttgaga gtacgtttcg atttctgact gtgttagcct ggaagtgctt gtcccaacct
     4801 tgtttctgag catgaacgcc cgcaagccaa catgttagtt gaagcatcag ggcgattagc
     4861 agcatgatat caaaacgctc tgagctgctc gttcggctat ggcgtaggcc tagtccgtag
     4921 gcaggacttt tcaagtctcg gaaggtttct tcaatctgca ttcgcttcga atagatatta
     4981 acaagttgtt tgggtgttcg aatttcaaca ggtaagttag ttgctagaac ccatggctcc
     5041 tttgccgacg ctgagtagat tttaggtgac gggtggtgac aatgagtccg tgtcgagcgc
     5101 tgattttttc ggcctttaga gcgagattta tacaatagaa tttggcatga gattggattg
     5161 cttttagtca gcctcttata gcctaaagtc tttgagtgac tagatgacat atcatgtaag
     5221 ttgctgatag gtttccagtt ttccgctcct aggtctgcat attgtacttt tcctcttact
     5281 cgacttaacc agtaccaacc cagcttctca acggatttat accatggcac tttaaagcca
     5341 gcatcactga caatgagcgg tgtggtgtta ctcggtagaa tgctcgcaag gtcggctaga
     5401 aattggtcat gagctttctt tgaacattgc tctgaaagcg ggaacgcttt ctcataaaga
     5461 gtaacagaac gaccgtgtag tgcgactgaa gctcgcaata ccataagtcg tttttgctca
     5521 cgaatatcag accagtcaac aagtacaatg ggcatcgtat tgcccgaaca gataaagcta
     5581 gcatgccaac ggtatacagc gagtcgctct ttgtggaggt gacgattacc taacaatcgg
     5641 tcgattcgtt tgatgttatg ttttgttctc gctttggttg gcaggttacg gccaagttcg
     5701 gtaagagtga gagttttaca gtcaagtaat gcgtggcaag ccaacgttaa gctgttgagt
     5761 cgttttaagt gtaattcggg gcagaattgg taaagagagt cgtgtaaaat atcgagttcg
     5821 cacatcttgt tgtctgatta ttgatttttc gcgaaaccat ttgatcatat gacaagatgt
     5881 gtatccacct taacttaatg atttttacca aaatcattag gggattcatc agtaccgagc
     5941 tcgaattcca cacattatat tgttatccgc tcacaatgtc aattcgccct atagtgagtc
     6001 gtattacaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     6061 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     6121 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga cgcgccctgt
     6181 agcggcgcat taagcgcggc ggg
//
