LOCUS       sequence-366648-        5222 bp    DNA     circular SYN 15-APR-2025
DEFINITION  For the mammalian expression of the tardigrade protein 
            CAHS2_PARRC_98-187. This is one of the bottom performing targets in 
            our apoptosis assay (it induced apoptosis)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5222)
  AUTHORS   Veling MT, Nguyen DT, Thadani NN, Oster ME, Rollins NJ, Brock KP, 
            Bethel NP, Lim S, Baker D, Way JC, Marks DS, Chang RL, Silver PA
  TITLE     Natural and Designed Proteins Inspired by Extremotolerant Organisms 
            Can Form Condensates and Attenuate Apoptosis in Human Cells.
  JOURNAL   ACS Synth Biol. 2022 Mar 18;11(3):1292-1302. doi: 
            10.1021/acssynbio.1c00572. Epub 2022 Feb 18.
  PUBMED    35176859
REFERENCE   2  (bases 1 to 5222)
  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"; doi: 
            "10.1021/acssynbio.1c00572"; journalName: "ACS Synth Biol"; date: 
            "2022-03-18- 18"; volume: "11"; issue: "3"; pages: "1292-1302"
COMMENT     Sequence Label: pST_CAHS2_PARRC_98-187 (pBS0278)
FEATURES             Location/Qualifiers
     source          1..5222
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(40..59)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        231..610
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        611..814
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     765..785
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     859..878
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        859..877
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      895..904
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1186..1209
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1226..1243)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1232..1456
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1502..1930
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1589..1608)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1799..1820
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1939..1959)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1944..2273
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2124..2259
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2186..2205
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2321..2368
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2387..2761
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    2891..3012
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2928..2947)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2982..3001
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3061..3077)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3061..3077)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3074..3096)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3085..3101
                     /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(3109..3115,3116..3133,3134..3139))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3292..3309)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3463..4051)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3543..3562)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4222..5013,5014..5082))
                     /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     4845..4864
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5083..5187)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc
       61 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
      121 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
      181 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt
      241 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
      301 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      361 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      421 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      481 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      541 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      601 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      661 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      721 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      781 gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc
      841 ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg gctagccacc
      901 atggccgcca agaccagaga gaacgagatg ctgggccagc agtacgagaa ggagctggag
      961 agaaagagcg aggcctacag aaagcaccag gaggtggagg ccgacaagat ccggaaggag
     1021 ctggagaagc agcacatgag agacatcgag ttccggaagg agatcgccga gctggccatc
     1081 gagaaccaga agcggatgat cgacctggag tgtcggtacg ctaagaagga tatggaccgg
     1141 gagagaacca aggtgcgtat gatgctggag cagtgaggat ctggggacta caaggacgac
     1201 gacgacaagt gaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     1261 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     1321 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     1381 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     1441 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     1501 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     1561 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     1621 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     1681 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     1741 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     1801 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     1861 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     1921 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     1981 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     2041 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     2101 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     2161 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     2221 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     2281 cgggagcttg tatatccatt ttcggatctg atcagcacgt gttgacaatt aatcatcggc
     2341 atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccatgg ccaagttgac
     2401 cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga
     2461 ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga
     2521 cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg
     2581 ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa
     2641 cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga
     2701 gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg
     2761 acacgtgcta cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     2821 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     2881 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     2941 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     3001 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     3061 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     3121 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactta cattaattgc
     3181 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     3241 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     3301 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     3361 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     3421 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     3481 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     3541 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     3601 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     3661 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3721 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3781 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3841 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3901 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     3961 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4021 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4081 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4141 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     4201 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     4261 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     4321 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     4381 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     4441 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     4501 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     4561 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     4621 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     4681 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     4741 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     4801 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     4861 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     4921 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     4981 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5041 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     5101 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     5161 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     5221 cg
//
