LOCUS       sequence-412565-        6620 bp    DNA     circular SYN 14-APR-2025
DEFINITION  To express C2m2-mKate in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6620)
  AUTHORS   Dirvelyte E, Bujanauskiene D, Jankaityte E, Daugelaviciene N, 
            Kisieliute U, Nagula I, Budvytyte R, Neniskyte U
  TITLE     Genetically encoded phosphatidylserine biosensor for in vitro, ex 
            vivo and in vivo labelling.
  JOURNAL   Cell Mol Biol Lett. 2023 Jul 27;28(1):59. doi: 
            10.1186/s11658-023-00472-7.
  PUBMED    37501184
REFERENCE   2  (bases 1 to 6620)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Mol 
            Biol Lett."; date: "2023-07-27"; volume: "28(1):59. doi"; pages: " 
            10.1186/s11658-023-00472-7"
COMMENT     Sequence Label: pET21a-C2m2-mKate
FEATURES             Location/Qualifiers
     source          1..6620
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     347..366
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     600..617
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    632..771
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(697..716)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     857..879
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(873..1064)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1873..2955)
                     /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     2917..2936
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2956..3033)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3239..3258
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3342..3361
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3342..3360
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3361..3385
                     /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             3400..3422
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3994..4686
                     /codon_start=1
                     /product="monomeric red fluorescent protein optimized for 
                     use as a fusion tag (Shemiakina et al., 2012)"
                     /label=FusionRed
                     /note="mammalian codon-optimized"
                     /translation="VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFMYGSKTFINHPQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPANGPVMQKKTLGWEASTETLYPADGGLEGACDMALK
                     LVGGGHLICNLETTYRSKKPAKNLKMPGVYNVDRRLERIKEADNETYVEQHEVAVARYS
                     TGGAGDGGK"
     CDS             4693..4710
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4763..4781)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4777..4824
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4861..5316
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4948..4967)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5158..5179
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5342..5446
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5447..5515,5516..6307)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5665..5684)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6478..6620,1..446)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
       61 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
      121 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
      181 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
      241 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
      301 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
      361 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
      421 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
      481 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
      541 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
      601 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
      661 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
      721 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
      781 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
      841 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
      901 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
      961 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     1021 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     1081 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     1141 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     1201 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     1261 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     1321 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     1381 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     1441 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     1501 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     1561 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     1621 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     1681 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     1741 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     1801 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     1861 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     1921 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     1981 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     2041 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     2101 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     2161 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     2221 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     2281 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     2341 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     2401 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     2461 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     2521 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     2581 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     2641 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     2701 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     2761 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     2821 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     2881 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     2941 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     3001 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     3061 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     3121 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     3181 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     3241 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     3301 tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg
     3361 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
     3421 gatatacata tgcacggatg ttctgagccc ctgggcctga agaataacac aattcctgac
     3481 agccagatgt cagcctccag cagctacaat acatggaacc tgcgtgcttt tggctggtac
     3541 ccccacttgg gaaggctgga taatcagggc aatatcaatg cctggacggc tcagagcaac
     3601 agtgccaagg aatggctgca ggttgacctg ggcactcaga ggcaagtgac aggaatcatc
     3661 acccaggggg cccgtgactt tggccacatc cagtatgtgg cgtcctacaa ggtagcccac
     3721 agtgatgatg gtgtgcagtg gactgtatat gaggagcaag gaagcagcaa ggtcttccag
     3781 ggcaacttgg acaacaactc ccacaagaag aacatcttcg agaaaccctt catggctcgc
     3841 tacgtgcgtg tccttccagt gtcctggcat aaccgcatca ccctgcgcct ggagctgctg
     3901 ggctgtaagc ttggcactgg aggctctgga ggcactggag gctctggagg caccggtagc
     3961 actagcggcg gaagcggcgg gacaggtacg cgtgtgagcg agctgattaa ggagaacatg
     4021 cacatgaagc tgtacatgga gggcaccgtg aacaaccacc acttcaagtg cacatccgag
     4081 ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggtcgt cgagggcggc
     4141 cctctcccct tcgccttcga catcctggct accagcttca tgtacggcag caaaaccttc
     4201 atcaaccacc ctcagggcat ccccgacttc tttaagcagt ccttccctga gggcttcaca
     4261 tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc
     4321 ctccaggacg gctgcctcat ctacaacgtc aagattagag gggtgaactt cccagccaac
     4381 ggccctgtga tgcagaagaa aacactcggc tgggaggcct ccaccgagac gctgtacccc
     4441 gctgacggcg gcctggaagg cgcatgtgac atggccctga agctcgtggg cgggggccac
     4501 ctgatctgca acttggagac cacatacaga tccaagaaac ccgctaagaa cctcaagatg
     4561 cccggcgtct acaacgtgga caggagactg gaaagaatca aggaggccga caatgagacc
     4621 tacgtcgagc agcacgaggt ggctgtggcc agatactcta ctggtggcgc tggtgatgga
     4681 ggtaaactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
     4741 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     4801 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     4861 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     4921 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     4981 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     5041 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     5101 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     5161 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     5221 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     5281 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     5341 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5401 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     5461 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5521 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5581 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     5641 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     5701 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     5761 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     5821 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     5881 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     5941 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc
     6001 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     6061 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     6121 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     6181 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     6241 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     6301 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     6361 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6421 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     6481 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     6541 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6601 cagagcgcag ataccaaata
//
