LOCUS       sequence-367387-        6436 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of rHCaMP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6436)
  AUTHORS   Wang L, Hiblot J, Popp C, Xue L, Johnsson K
  TITLE     Environmentally Sensitive Color-Shifting Fluorophores for 
            Bioimaging.
  JOURNAL   Angew Chem Int Ed Engl. 2020 Dec 1;59(49):21880-21884. doi: 
            10.1002/anie.202008357. Epub 2020 Sep 28.
  PUBMED    32762146
REFERENCE   2  (bases 1 to 6436)
  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: 
            "10.1002/anie.202008357"; journalName: "Angew Chem Int Ed Engl"; 
            date: "2020-12-1- 1"; volume: "59"; issue: "49"; pages: 
            "21880-21884"
COMMENT     Sequence Label: pCDNA5/FRT_rHCaMP
FEATURES             Location/Qualifiers
     source          1..6436
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(30..47)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(201..789)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(281..300)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(960..1751,1752..1820))
                     /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     1583..1602
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1821..1925)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2000..2019)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2191..2570
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2571..2774
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2725..2745
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2819..2838
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2819..2837
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3806..3862
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     primer_bind     complement(4344..4361)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4350..4574
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4707..4726)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4858..4905
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             4913..5935
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4913..5935
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    6065..6186
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6102..6121)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6156..6175
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6235..6251)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6235..6251)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6248..6270)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6259..6275
                     /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(6283..6289,6290..6307,6308..6313))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6328..6349
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
       61 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
      121 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
      181 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
      241 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
      301 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
      361 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
      421 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
      481 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
      541 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
      601 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
      661 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
      721 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
      781 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
      841 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
      901 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
      961 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     1021 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     1081 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     1141 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     1201 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     1261 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     1321 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     1381 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     1441 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     1501 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     1561 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     1621 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     1681 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     1741 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     1801 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     1861 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     1921 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
     1981 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
     2041 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
     2101 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
     2161 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
     2221 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
     2281 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
     2341 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
     2401 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
     2461 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
     2521 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
     2581 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
     2641 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
     2701 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
     2761 atataagcag agctctctgg ctaactagag aacccactgc ttactggctt atcgaaatta
     2821 atacgactca ctatagggag acccaagctg gctagcgttt aaacttacca ccatgggatc
     2881 cgaaatcggt actggctttc cattcgaccc ccattatgtg gaagtcctgg gcgagcgcat
     2941 gcactacgtc gatgttggtc cgcgcgatgg cacccctgtg ctgttcctgc acggtaaccc
     3001 gacctcctcc tacgtgtggc gcaacatcat cccgcatgtt gcaccgaccc atcgctgcat
     3061 tgctccagac ctgatcggta tgggcaaatc cgacaaacca gacctgggtt atttcttcga
     3121 cgaccacgtc cgcttcatgg atgccttcat cgaagccctg ggtctggaag aggtcgtcct
     3181 ggtcattcac gactggggct ccgctctggg tttccactgg gccaagcgca atccagagcg
     3241 cgtcaaaggt attgcattta tggagttcat ccgccctatc ccgacctggg acgaatggcc
     3301 agaatttgcc cgcgagacct tccagctgcc ggaccaactg actgaagagc agatcgcaga
     3361 atttaaagag gaattctccc tatttgacaa ggacggggat gggacaataa caaccaagga
     3421 gctggggacg gtgatgcggt ctctggggca gaaccccaca gaagcagagc tgcaggacat
     3481 gatcaatgaa gtagatgccg acggtgacgg cacaatcgac ttccctgagt tcctgacaat
     3541 gatggcaaga aaaatgaaat acagggacac ggaagaagaa attagagaag cgttcggtgt
     3601 gtttgataag gatggcaatg gctacatcag tgcagcagag cttcgccacg tgatgacaaa
     3661 ccttggagag aagttaacag atgaagaggt tgatgaaatg atcagggaag cagacatcga
     3721 tggggatggt caggtaaact acgaagagtt tgtacaaatg atgacagcga agggtggcac
     3781 cggtggctct atggtcgact catcacgtcg taagtggaat aagacaggtc acgcagtcag
     3841 agctataggt cggctgagct cactcgaggc cttccgcacc accgacgtcg gccgcaagct
     3901 gatcatcgat cagaacgttt ttatcgaggg tacgctgccg atgggtgtcg tccgcccgct
     3961 gactgaagtc gagatggacc attaccgcga gccgttcctg aatcctgttg accgcgagcc
     4021 actgtggcgc ttcccaaacg agctgccaat cgccggtgag ccagcgaaca tcgtcgcgct
     4081 ggtcgaagaa tacatggact ggctgcacca gtcccctgtc ccgaagctgc tgttctgggg
     4141 caccccaggc gttctgatcc caccggccga agccgctcgc ctggccaaaa gcctgcctaa
     4201 ctgcaaggct gtggacatcg gcccgggtct gaatctgctg caagaagaca acccggacct
     4261 gatcggcagc gagatcgcgc gctggctgtc tactctggag atttccggtt agtctagagg
     4321 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     4381 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     4441 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     4501 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     4561 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     4621 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     4681 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     4741 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     4801 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtacctagaa
     4861 gttcctattc cgaagttcct attctctaga aagtatagga acttccttgg ccaaaaagcc
     4921 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga
     4981 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
     5041 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     5101 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag
     5161 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     5221 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc
     5281 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     5341 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     5401 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     5461 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     5521 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     5581 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     5641 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     5701 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     5761 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     5821 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     5881 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagcacgt
     5941 actacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     6001 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     6061 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     6121 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     6181 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     6241 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     6301 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     6361 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     6421 acgcgcgggg agaggc
//
