LOCUS       sequence-461205-        8080 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Low expression plasmid of STIM1 with fluorescent calcium biosensor 
            CaMPARI2 for Matreyek Bxb1(GT) landing pad.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8080)
  TITLE     Comprehensive mutational characterization of the calcium-sensing 
            STIM1 EF-hand reveals residues essential for structure and function
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8080)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: J364B_AttB_mkozak_STIM1_IRES_CaMPARI2-P2A-PuroR
FEATURES             Location/Qualifiers
     source          1..8080
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1162..1617)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1299..1320)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1511..1530
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1744..1766
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1758..1775
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1759..1775
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1782..1801
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1782..1800
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1874..1911
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     misc_feature    join(4121..4695,4696..4698,4699..4707)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4288..4305)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4515..4534
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4771..4788
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4795..5235
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEA
                     ELQDMINEVDADGDGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAEL
                     RHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVVMMTAK"
     CDS             6031..6054
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6073..6099
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6118..6147
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             6160..6216
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     6710..6730
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6921..7042
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6958..6977)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7012..7031
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        complement(7051..7069)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7090..7106)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7090..7106)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7103..7125)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7114..7130
                     /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(join(7138..7144,7145..7162,7163..7168))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7183..7204
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7321..7338)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7492..8080)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7572..7591)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     1201 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     1261 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     1321 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     1381 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     1441 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     1501 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     1561 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
     1621 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     1681 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     1741 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac tcactatagg
     1801 gcgaattggg taccgggccc cccctcgagg tcgacgatgt aggtcacggc attccggact
     1861 cagcgctcga gccggcttgt cgacgacggc ggtctccgtc gtcaggatca tccgcagatt
     1921 ataccgcaac tacaccattg tatggatgta tgcgtccgtc ttgccctgtg gctcctctgg
     1981 ggactcctcc tgcaccaggg ccagagcctc agccatagtc acagtgagaa ggcgacagga
     2041 accagctcgg gggccaactc tgaggagtcc actgcagcag agttttgccg aattgacaag
     2101 cccctgtgtc acagtgagga tgagaaactc agcttcgagg cagtccgtaa catccacaaa
     2161 ctgatggacg atgatgccaa tggtgatgtg gatgtggaag aaagtgatga gttcctgagg
     2221 gaagacctca attaccatga cccaacagtg aaacacagca ccttccatgg tgaggataag
     2281 ctcatcagcg tggaggacct gtggaaggca tggaagtcat cagaagtata caattggacc
     2341 gtggatgagg tggtacagtg gctgatcaca tatgtggagc tgcctcagta tgaggagacc
     2401 ttccggaagc tgcagctcag tggccatgcc atgccaaggc tggctgtcac caacaccacc
     2461 atgacaggga ctgtgctgaa gatgacagac cggagtcatc ggcagaagct gcagctgaag
     2521 gctctggaca ctgtgctctt tgggcctcct ctcttgactc gccataatca cctcaaggac
     2581 ttcatgctgg tggtgtctat cgttattggt gtgggcggct gctggtttgc ctatatccag
     2641 aaccgttact ccaaggagca catgaagaag atgatgaagg acttggaggg gttacaccga
     2701 gctgagcaga gtctgcatga ccttcaggaa aggctgcaca aggcccagga ggagcaccgc
     2761 acagtggagg tggagaaggt ccatctggaa aagaagctgc gcgatgagat caaccttgct
     2821 aagcaggaag cccagcggct gaaggagctg cgggagggta ctgagaatga gcggagccgc
     2881 caaaaatatg ctgaggagga gttggagcag gttcgggagg ccttgaggaa agcagagaag
     2941 gagctagaat ctcacagctc atggtatgct ccagaggccc ttcagaagtg gctgcagctg
     3001 acacatgagg tggaggtgca atattacaac atcaagaagc aaaatgctga gaagcagctg
     3061 ctggtggcca aggagggggc tgagaagata aaaaagaaga gaaacacact ctttggcacc
     3121 ttccacgtgg cccacagctc ttccctggat gatgtagatc ataaaattct aacagctaag
     3181 caagcactga gcgaggtgac agcagcattg cgggagcgcc tgcaccgctg gcaacagatc
     3241 gagatcctct gtggcttcca gattgtcaac aaccctggca tccactcact ggtggctgcc
     3301 ctcaacatag accccagctg gatgggcagt acacgcccca accctgctca cttcatcatg
     3361 actgacgacg tggatgacat ggatgaggag attgtgtctc ccttgtccat gcagtcccct
     3421 agcctgcaga gcagtgttcg gcagcgcctg acggagccac agcatggcct gggatctcag
     3481 agggatttga cccattccga ttcggagtcc tccctccaca tgagtgaccg ccagcgtgtg
     3541 gcccccaaac ctcctcagat gagccgtgct gcagacgagg ctctcaatgc catgacttcc
     3601 aatggcagcc accggctgat cgagggggtc cacccagggt ctctggtgga gaaactgcct
     3661 gacagccctg ccctggccaa gaaggcatta ctggcgctga accatgggct ggacaaggcc
     3721 cacagcctga tggagctgag cccctcagcc ccacctggtg gctctccaca tttggattct
     3781 tcccgttctc acagccccag ctccccagac ccagacacac catctccagt tggggacagc
     3841 cgagccctgc aagccagccg aaacacacgc attccccacc tggctggcaa gaaggctgtg
     3901 gctgaggagg ataatggctc tattggcgag gaaacagact ccagcccagg ccggaagaag
     3961 tttcccctca aaatctttaa gaagcctctt aagaagtagt aacttaagaa ttcaccggtc
     4021 tgacctcctt ctccctctct tcaggtctgc agtcgacggt accgcgggcc cgggatccac
     4081 cggatctaga taactgatca ctgtggaatc agagagactg cccctctccc tctccccccc
     4141 ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat
     4201 tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct
     4261 tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg
     4321 tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct gtagcgaccc
     4381 tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg
     4441 tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg
     4501 tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga
     4561 aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt
     4621 agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa
     4681 aaacacgatg ataatatggc cacaaccatg ctgcagaacg agcttgctct taagttggct
     4741 ggacttgata ttaacaagac tggaggttct catcatcacc accaccatgg atccgaccaa
     4801 ctgactgaag agcagatcgc agaatttaaa gaggctttct ccctatttga caaggacggg
     4861 gatgggacaa taacaaccaa ggagctgggg acggtgatgc ggtctctggg gcagaacccc
     4921 acagaagcag agctgcagga catgatcaat gaagtagatg ccgacggtga cggcacaatc
     4981 gacttccctg agttcctgac aatgatggca agaaaaatga aagacacaga cagtgaagaa
     5041 gaaattagag aagcgttccg tgtgtttgat aaggatggca atggctacat cagtgcagca
     5101 gagcttcgcc acgtgatgac aaaccttgga gagaagttaa cagatgaaga ggttgatgaa
     5161 atgatcaggg aagcagacat cgatggggat ggtcaggtaa actacgaaga gtttgtagtg
     5221 atgatgacag cgaagctcga gtgcgagaaa atctatgtgc gtgatggagt gctgacgggt
     5281 gatattcata tgatcttgtt gcttgaagga aatgcccatt accgatgtga cttcagaact
     5341 acttacaaag ctaaggagaa gggtgtcaag ttaccaggcg tgcactatgt ggaccacacc
     5401 attgagattt taagccatga caaagattac aacaaggtta agatctatga gtatgctgtt
     5461 gctcattctg gattgcctga caatgccaga cgaggcggta ccggcggatc catggtgagt
     5521 gcgattaagc cagacatgaa gatcaaactg cgtatggaag gcaacgtaaa cgggcaccac
     5581 tttgtgatcg acggagatgg tacaggcaag ccttatgagg gaaaacagac catggatctt
     5641 gaagtcaaag agggcggacc tctgcctttt gcctttgata tcctgaccac tgcattccat
     5701 tacggcaaca gggtattcgt gaaatatcca gacaacatac aagactattt taagcagtcg
     5761 tttcctaagg ggtattcgtg ggaacgaagc atgactttcg aagacggggg catttgctat
     5821 gccagaagcg acataacaat ggaaggggac actttctata ataaagttcg attttatggt
     5881 accaactttc ccgccaatgg tccagttatg cagaagaaga cgctgaaatg gatgccgagc
     5941 tggacgcgtt catcacgtcg taagttcaat aagacaggtc acgcactgag agctataggt
     6001 cggctgagct caggaggttc tggaggttct gattacaagg acgacgatga caagggaggt
     6061 tctggaggtt cttacccata cgacgtacca gattacgctg gaggttctgg aggttctgag
     6121 cagaagctga tctcagagga ggacctgcgt acgagcggag ctactaactt cagcctgctg
     6181 aagcaggctg gagacgtgga ggagaaccct ggacctaccg agtacaagcc cacggtgcgc
     6241 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     6301 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     6361 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcagacgac
     6421 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     6481 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     6541 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     6601 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     6661 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     6721 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     6781 acctggtgca tgacccgcaa gtaataggga tcctgatcat aatcagccat accacatttg
     6841 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa
     6901 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca
     6961 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     7021 ccaaactcat caatgtatct taggagatcc cctttagtga gggttaattt cgagcttggc
     7081 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     7141 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     7201 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     7261 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     7321 ctcgctcact gactcgctgc actcggtcgt tcggctgcgg cgagcggtat cagctcactc
     7381 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     7441 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     7501 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     7561 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     7621 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     7681 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     7741 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     7801 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     7861 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     7921 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     7981 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     8041 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
//
