LOCUS       sequence-180071-        7422 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ptrc-ECFP-KaiC fusion cloned into Km resistance gene of pACYC177. To
            look for KaiC localization in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7422)
  AUTHORS   Cohen SE, Erb ML, Selimkhanov J, Dong G, Hasty J, Pogliano J, Golden
            SS
  TITLE     Dynamic localization of the cyanobacterial circadian clock proteins.
  JOURNAL   Curr Biol. 2014 Aug 18;24(16):1836-44. doi: 
            10.1016/j.cub.2014.07.036. Epub 2014 Aug 7.
  PUBMED    25127213
REFERENCE   2  (bases 1 to 7422)
  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: 
            "10.1016/j.cub.2014.07.036"; journalName: "Curr Biol"; date: 
            "2014-08-18- 18"; volume: "24"; issue: "16"; pages: "1836-44"
COMMENT     Sequence Label: pAM5084
FEATURES             Location/Qualifiers
     source          1..7422
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..546
                     /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     447..466
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     663..680
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(1130..1149)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(1205..2287)
                     /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     2249..2268
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2288..2365)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     promoter        join(2433..2438,2439..2455,2456..2462)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2470..2486
                     /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             join(2522..2524,2525..2527,2528..3238)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2567..2588)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2828..2847)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3175..3196
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3239..3268
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     terminator      4873..4900
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(6207..6225)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6293..6397
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6398..6466,6467..7258)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6616..6635)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcgt tttggtctgc gcgtaatctc ttgctctgaa aacgaaaaaa ccgccttgca
       61 gggcggtttt tcgaaggttc tctgagctac caactctttg aaccgaggta actggcttgg
      121 aggagcgcag tcaccaaaac ttgtcctttc agtttagcct taaccggcgc atgacttcaa
      181 gactaactcc tctaaatcaa ttaccagtgg ctgctgccag tggtgctttt gcatgtcttt
      241 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcggac tgaacggggg
      301 gttcgtgcat acagtccagc ttggagcgaa ctgcctaccc ggaactgagt gtcaggcgtg
      361 gaatgagaca aacgcggcca taacagcgga atgacaccgg taaaccgaaa ggcaggaaca
      421 ggagagcgca cgagggagcc gccaggggga aacgcctggt atctttatag tcctgtcggg
      481 tttcgccacc actgatttga gcgtcagatt tcgtgatgct tgtcaggggg gcggagccta
      541 tggaaaaacg gctttgccgc ggccctctca cttccctgtt aagtatcttc ctggcatctt
      601 ccaggaaatc tccgccccgt tcgtaagcca tttccgctcg ccgcagtcga acgaccgagc
      661 gtagcgagtc agtgagcgag gaagcggaat atatcctgta tcacatattc tgctgacgca
      721 ccggtgcagc cttttttctc ctgccacatg aagcacttca ctgacaccct catcagtgcc
      781 aacatagtaa gccagtatac actccgctag cgctgaggtc tgcctcgtga agaaggtgtt
      841 gctgactcat accaggcctg aatcgcccca tcatccagcc agaaagtgag ggagccacgg
      901 ttgatgagag ctttgttgta ggtggaccag ttggtgattt tgaacttttg ctttgccacg
      961 gaacggtctg cgttgtcggg aagatgcgtg atctgatcct tcaactcagc aaaagttcga
     1021 tttattcaac aaagccacgt tgtgtctcaa aatctctgat gttacattgc acaagataaa
     1081 aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt
     1141 atgagccata ttcaacggga aacgtcttgc tcgagcaggt agtcggccaa taacccaggg
     1201 attttcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     1261 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     1321 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     1381 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     1441 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     1501 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     1561 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     1621 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     1681 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     1741 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     1801 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     1861 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     1921 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     1981 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     2041 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     2101 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     2161 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     2221 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     2281 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     2341 aggttttgca ccattcgatg gtgtcaacgt aaatgcatgc cgcttcgcct tcgcgcgcga
     2401 attgatctgc ggcggccgcc tgaaatgagc tgttgacaat taatcatccg gctcgtataa
     2461 tgtgtggaat tgtgagcgga taacaatttc acacgcatgc ggatcctaag gaggaaaaaa
     2521 aatggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     2581 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     2641 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     2701 cctcgtgacc accctgacct ggggcgtgca gtgcttcagc cgctaccccg accacatgaa
     2761 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     2821 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     2881 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     2941 caagctggag tacaactaca tcagccacaa cgtctatatc accgccgaca agcagaagaa
     3001 cggcatcaag gccaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     3061 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     3121 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     3181 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagga
     3241 acagaagctt atcagcgaag aagatctggg ctctggatcg ggttcaggaa tgacttccgc
     3301 tgagatgact agccctaata ataattctga gcaccaagcc atcgctaaga tgcgcacgat
     3361 gattgaaggc tttgatgata ttagtcatgg cggtcttcca atcgggcgat cgaccctcgt
     3421 tagtggtact tcaggaaccg gcaagaccct tttttctatt caatttctct ataacggtat
     3481 tatcgagttt gatgagcctg gggttttcgt tactttcgaa gagaccccgc aagatatcat
     3541 taaaaacgcc cgtagttttg gctgggattt agccaagctg gtcgatgagg gcaaactatt
     3601 tattcttgat gcttcacccg atccagaagg tcaagaggtt gttggcggct tcgatctctc
     3661 tgctctgatt gagcggatta attatgcaat tcaaaagtat cgagcgcggc gggtttcaat
     3721 tgactcggtc acgtccgttt tccagcaata tgatgcctct tctgtggttc gccgcgaact
     3781 ctttcggttg gtagctcgcc taaaacaaat tggggcaact acggtcatga ccaccgagcg
     3841 tatcgaggaa tatggcccga tcgctcgtta cggtgttgag gaatttgtct ccgataacgt
     3901 cgtgattctc cgcaacgttt tggaagggga gcgccgtcgc cgcaccctcg aaatcctcaa
     3961 gctacgtggc accagccaca tgaaagggga atatccgttc acgattacgg atcatggcat
     4021 caatatcttc ccgctcgggg caatgcgcct tacgcagcga tcgtcgaacg tgcgtgtttc
     4081 atctggtgtc gtccgactcg atgaaatgtg tggtgggggc ttctttaagg actcaatcat
     4141 tctggcaact ggcgctacag gcactggtaa aactctgtta gttagccgtt tcgttgagaa
     4201 tgcttgtgct aacaaagagc gggcgattct gttcgcttat gaagagtcac gagctcagct
     4261 gctccgcaat gcctattcat ggggaatgga ctttgaggag atggagcgcc aaaacctcct
     4321 caaaattgtt tgcgcctatc ctgaatctgc aggtcttgaa gaccatttgc agattattaa
     4381 gtcggagatc aatgacttta agccagctcg tattgcaatc gactccctct ctgctttggc
     4441 gcggggcgtt agcaacaatg ccttccgcca atttgtaatt ggtgtcactg gctacgcgaa
     4501 acaagaagaa atcacgggac tattcacaaa taccagtgat caatttatgg gagcgcattc
     4561 gattactgac tcccatatct caacaattac ggatacgatt atcttgctcc aatacgtcga
     4621 gattcgtggc gaaatgtccc gcgccattaa cgtcttcaag atgcgcggat cttggcatga
     4681 caaagcaatc cgcgaattca tgatcagcga caaagggccg gacatcaagg attctttccg
     4741 gaactttgag cggattattt caggttcgcc aacacggatt accgtcgatg agaaaagcga
     4801 actctcgcga attgtgcgcg gcgttcaaga aaaagggccg gagagctaga aatgaaggag
     4861 ctccccggca ggagaaggcc atcctgacgg atggcctttt tgaagctttc tagaatcggg
     4921 ggcccccggg aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat
     4981 tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc
     5041 ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt
     5101 ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa
     5161 agaaatgcat aagcttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc
     5221 acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt
     5281 cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc
     5341 tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa
     5401 attgcagttt catttgatgc tcgatgagtt tttctaatca gaattggtta attggttgta
     5461 acactggcag agcattacgc tgacttgacg ggacggcggc tttgttgaat aaatcgaact
     5521 tttgctgagt tgaaggatca gatcacgcat cttcccgaca acgcagaccg ttccgtggca
     5581 aagcaaaagt tcaaaatcac caactggtcc acctacaaca aagctctcat caaccgtggc
     5641 tccctcactt tctggctgga tgatggggcg attcaggcct ggtatgagtc agcaacacct
     5701 tcttcacgag gcagacctca gcgctcaaag atgcaggggt aaaagctaac cgcatcttta
     5761 ccgacaaggc atccggcagt tcaacagatc gggaagggct ggatttgctg aggatgaagg
     5821 tggaggaagg tgatgtcatt ctggtgaaga agctcgaccg tcttggccgc gacaccgccg
     5881 acatgatcca actgataaaa gagtttgatg ctcagggtgt agcggttcgg tttattgacg
     5941 acgggatcag taccgacggt gatatggggc aaatggtggt caccatcctg tcggctgtgg
     6001 cacaggctga acgccggagg atcctagagc gcacgaatga gggccgacag gaagcaaagc
     6061 tgaaaggaat caaatttggc cgcaggcgta ccgtggacag gaacgtcgtg ctgacgcttc
     6121 atcagaaggg cactggtgca acggaaattg ctcatcagct cagtattgcc cgctccacgg
     6181 tttataaaat tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     6241 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     6301 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     6361 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     6421 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     6481 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     6541 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     6601 cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca
     6661 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     6721 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     6781 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     6841 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     6901 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt
     6961 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     7021 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     7081 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     7141 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     7201 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     7261 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     7321 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     7381 ttcgttccac tgagcgtcag accccttaat aagatgatct tc
//
