LOCUS       sequence-197246-        3804 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses KcsA-Kv1.3 protein with a C-terminal His tag and a deGFP 
            fluorescent protein with a different ribosome binding site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3804)
  AUTHORS   Guo S, Vaish A, Chen Q, Murray RM
  TITLE     Expressing Biologically Active Membrane Proteins in a Cell-Free 
            Transcription-Translation Platform
  JOURNAL   bioRxiv 104455
REFERENCE   2  (bases 1 to 3804)
  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"; journalName: "bioRxiv 
            104455"
COMMENT     Sequence Label: pSG69-KcsA-Kv1.3-deGFP
FEATURES             Location/Qualifiers
     source          1..3804
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             7..29
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             531..548
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(668..689)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(929..948)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1276..1297
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     rep_origin      complement(1663..2251)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1743..1762)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2422..3213,3214..3282))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLHVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRHEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHATRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3045..3064
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3283..3387)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3455..3473
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 aataattttg tttaacttta agaaggagat atacaatgga cgaagttgat ggtccgccaa
       61 tgttgtccgg tctgcttgct cgtctggtta agttgcttct gggccgtcac ggttctgcgc
      121 tgcactggcg tgctgcaggc gctgccaccg tccttctggt gatcgtactg cttgctggtt
      181 cctacttggc ggttctggcg gaagcggatg atccgaccag cggctttagc agcattccgg
      241 acgccttgtg gtggtctgtc gataccgcta ctaccgtggg ttacggcgac ttgtatccgg
      301 tgaccctttg gggtcgtctg gtagcggttg tcgttatggt ggctggcatc acctccttcg
      361 gtctggtaac tgctgccctt gctacttggt ttgtaggccg cgaacaagag cgtcgtggtc
      421 atttcgttcg tcactctgaa aaagcggctg aggaggcgta cacccgtact acccgtgctc
      481 tgcacgagcg ctttgaccgt ctggaacgta tgcttgacga taaccgtcgt catcatcatc
      541 atcatcattg aagggcccaa gttcacttaa aaaggagatc aacaatgaaa gcaattttcg
      601 tactgaaaca tcttaatcat gcaatggagg ctttctaatg gagcttttca ctggcgttgt
      661 tcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
      721 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
      781 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
      841 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
      901 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
      961 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     1021 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     1081 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     1141 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     1201 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     1261 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tctaactcga
     1321 gcaaagcccg ccgaaaggcg ggcttttctg tgtcgaccga tgcccttgag agccttcaac
     1381 ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtg
     1441 ttctttatca tgcaactcgt aggacaggtg ccggcagctc tcttccgctt cctcgctcac
     1501 ttactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     1561 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     1621 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     1681 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     1741 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     1801 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     1861 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     1921 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     1981 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     2041 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     2101 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     2161 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     2221 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     2281 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     2341 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     2401 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     2461 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     2521 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgtgacccac gctcaccggc
     2581 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     2641 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     2701 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     2761 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttgcatgatc
     2821 ccccatgttg tgcaaaaaag ctgttagctc cttcggtcct ccgatcgttg tcagaagtaa
     2881 gttggccgca gtgttatcac tcattgttat ggcagcactg cataattctc ttactgtcat
     2941 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     3001 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata ccgcgccaca
     3061 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcgtggcgaa aactctcaag
     3121 gatcttaccg ctgtttagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     3181 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acatgaaggc aaaatgccgc
     3241 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     3301 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     3361 gaaaaataaa caaataggtg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     3421 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     3481 tgttcaagaa ttctggcgaa tcctcttacc agccagaaaa cgacctttct gtggtgaaac
     3541 cggatgctgc aattcagagc gccagcaagt gggggacagc agatgacctg accgccgcag
     3601 agtggatgtt tgacatggtg atgactatcg caccatcagc cagaaaaccg aattttgctg
     3661 ggtgggctaa cgatatccgc ctgatgcgtg aacgtgacgg acgtaaccac cgcgacatgt
     3721 gtgtgctgtt ccgctgggca tgctgagcta acaccgtgcg tgttgacaat tttacctctg
     3781 gcggtgataa tggttgcagc tagc
//
