LOCUS       sequence-453692-        7160 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expressed a FRET sensor for determining proteolytic cleavage of the 
            N-terminal side of Catestatin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7160)
  TITLE     Chromogranin A constructs for mammalian expression
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7160)
  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            "Unpublished"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: pcDNA3.1-FRET_CST-N
FEATURES             Location/Qualifiers
     source          1..7160
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(1268..1270,1271..1981)
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1571..1590)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2154..2172)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(2751..2768)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2757..2981
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3027..3455
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3114..3133)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3324..3345
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3464..3484)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3469..3798
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3649..3784
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3711..3730
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3865..4659
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3919..3938)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4529..4548
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4833..4954
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4870..4889)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4924..4943
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5003..5019)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5003..5019)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5016..5038)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5027..5043
                     /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(5051..5057,5058..5075,5076..5081))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5096..5117
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5234..5251)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5405..5993)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5485..5504)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6164..6955,6956..7024))
                     /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     6787..6806
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7025..7129)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttgcca ccatgcgcag cgccgccgtg ctggccctgc tgctgtgcgc
      961 cggccaggtg accgccctgc ccgtgaacag ccccatgaac aagggcgaca ccgaggtgat
     1021 gaagtgcatc gtggaggtga tcagcgacac cctgagcaag cccagcccca tgcccgtgag
     1081 ccaggagtgc ttcgagaccc tgcgcggcga cgagcgcatc ctgagcatcc tgcgccacca
     1141 gaacctgctg aaggagctgc aggacctggc cctgcagggc gccaaggagc gcgcccacca
     1201 gcagaagaag cacagcggct tcgaggacga gctgagcgag gtgctggaga accagagcag
     1261 ccaggccgtg agcaagggcg aggagctgtt caccggcgtg gtgcccatcc tggtggagct
     1321 ggacggcgac gtgaacggcc acaagttcag cgtgagcggc gagggcgagg gcgacgccac
     1381 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     1441 caccctggtg accaccttcg gctacggcct gatgtgcttc gcccgctacc ccgaccacat
     1501 gaagcagcac gacttcttca agagcgccat gcccgagggc tacgtgcagg agcgcaccat
     1561 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     1621 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1681 ccacaagctg gagtacaact acaacagcca caacgtgtac atcatggccg acaagcagaa
     1741 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1801 ggccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1861 ccactacctg agctaccaga gcaagctgag caaggacccc aacgagaagc gcgaccacat
     1921 ggtgctgctg gagttcgtga ccgccgccgg catcaccctg ggcatggacg agctgtacaa
     1981 ggaattcgag gacaaccgcg acagcagcat gaagctggga tccgtgagca agggcgaggc
     2041 cgtgatcaag gagttcatgc gcttcaaggt gcacatggag ggcagcatga acggccacga
     2101 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
     2161 gaaggtgacc aagggcggcc ccctgccctt cagctgggac atcctgagcc cccagttcat
     2221 gtacggcagc cgcgccttca tcaagcaccc cgccgacatc cccgactact acaagcagag
     2281 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgccgtgac
     2341 cgtgacccag gacaccagcc tggaggacgg caccctgatc tacaaggtga agctgcgcgg
     2401 caccaacttc ccccccgacg gccccgtgat gcagaagaag accatgggct gggaggccag
     2461 caccgagcgc ctgtaccccg aggacggcgt gctgaagggc gacatcaaga tggccctgcg
     2521 cctgaaggac ggcggccgct acctggccga cttcaagacc acctacaagg ccaagaagcc
     2581 cgtgcagatg cccggcgcct acaacgtgga ccgcaagctg gacatcacca gccacaacga
     2641 ggactacacc gtggtggagc agtacgagcg cagcgagggc cgccacagca ccggcggcat
     2701 ggacgagctg tacaagtagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     2761 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     2821 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     2881 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     2941 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     3001 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     3061 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3121 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3181 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3241 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3301 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3361 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3421 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     3481 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3541 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3601 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     3661 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     3721 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     3781 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
     3841 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     3901 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     3961 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     4021 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     4081 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     4141 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     4201 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     4261 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     4321 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     4381 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     4441 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     4501 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     4561 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     4621 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     4681 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     4741 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     4801 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     4861 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4921 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     4981 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     5041 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     5101 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5161 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5221 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     5281 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     5341 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     5401 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5461 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5521 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5581 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5641 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5701 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5761 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5821 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5881 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5941 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     6001 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     6061 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     6121 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     6181 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     6241 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     6301 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     6361 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6421 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6481 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6541 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6601 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6661 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6721 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6781 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6841 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6901 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6961 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     7021 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     7081 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     7141 gaaaagtgcc acctgacgtc
//
