LOCUS       sequence-225435-        4863 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Selection for in-frame fusion expression constructs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4863)
  AUTHORS   McGeachy AM, Meacham ZA, Ingolia NT
  TITLE     An Accessible Continuous-Culture Turbidostat for Pooled Analysis of 
            Complex Libraries.
  JOURNAL   ACS Synth Biol. 2019 Apr 19;8(4):844-856. doi: 
            10.1021/acssynbio.8b00529. Epub 2019 Apr 2.
  PUBMED    30908907
REFERENCE   2  (bases 1 to 4863)
  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: 
            "10.1021/acssynbio.8b00529"; journalName: "ACS Synth Biol"; date: 
            "2019-04-19- 19"; volume: "8"; issue: "4"; pages: "844-856"
COMMENT     Sequence Label: pPGK1-(AscI)-lambda-N-T2A-SpHIS5 (pNTI347)
FEATURES             Location/Qualifiers
     source          1..4863
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     163..181
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(219..241)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     341..360
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        441..459
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     441..458
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    503..1006
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     CDS             1883..1945
                     /codon_start=1
                     /product="N-terminal RNA-binding domain from the lambda 
                     bacteriophage antiterminator protein N (Baron-Benhamou et 
                     al., 2004)"
                     /label=lambda N peptide
                     /translation="DAQTRRRERRAEKQAQWKAAN"
     CDS             1946..1999
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2000..2650
                     /codon_start=1
                     /gene="Schizosaccharomyces pombe his5"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=S. pombe his5
                     /note="yeast auxotrophic marker; corresponds to S. 
                     cerevisiae HIS3"
                     /translation="GRRAFVERNTNETKISVAIALDKAPLPEESNFIDELITSKHTNQK
                     GEQVIQVDTGIGFLDHMYHALAKHAGWSLRLYSRGDLIIDDHHTAEDTAIALGIAFKQA
                     MSNFAGVKRFGHAYCPLDEALSRSVVDLSGRPYAVIDLGLKREKVGELSCEMIPHLLYS
                     FSVAAGITLHVTCLYGSNDHHRAESAFKSLAVAMRAATSLTGSSEVPSTKGVL"
     terminator      2656..2853
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2957..2976)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2958..2976)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3063..3080)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3234..3822)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3314..3333)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3993..4784,4785..4853))
                     /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     4616..4635
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(4854..4863,1..95))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
       61 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
      121 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
      181 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
      241 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
      301 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
      361 ctgagagtgc accatatgga catattgtcg ttagaacgcg gctacaatta atacataacc
      421 ttatgtatca tacacatacg atttaggtga cactatagaa cgcggccgcc agctgaagct
      481 tcgtacgctg caggtccttt tcatcacgtg ctataaaaat aattataatt taaatttttt
      541 aatataaata tataaattaa aaatagaaag taaaaaaaga aattaaagaa aaaatagttt
      601 ttgttttccg aagatgtaaa agactctagg gggatcgcca acaaatacta ccttttatct
      661 tgctcttcct gctctcaggt attaatgccg aattgtttca tcttgtctgt gtagaagacc
      721 acacacgaaa atcctgtgat tttacatttt acttatcgtt aatcgaatgt atatctattt
      781 aatctgcttt tcttgtctaa taaatatata tgtaaagtac gctttttgtt gaaatttttt
      841 aaacctttgt ttattttttt ttcttcattc cgtaactctt ctaccttctt tatttacttt
      901 ctaaaatcca aatacaaaac ataaaaataa ataaacacag agtaaattcc caaattattc
      961 catcattaaa agatacgagg cgcgtgtaag ttacaggcaa gcgatccgtc cggatcctgt
     1021 agccctagac ttgatagcca tgacttcaac tcaagacgca cagatattat aacatctgca
     1081 taataggcat ttgcaagaat tactcgtgag taaggaaaga gtgaggaact atcgcatacc
     1141 tgcatttaaa gatgccgatt tgggcgcgaa tcctttattt tggcttcacc ctcatactat
     1201 tatcagggcc agaaaaagga agtgtttccc tccttcttga attgatgtta ccctcataaa
     1261 gcacgtggcc tcttatcgag aaagaaatta ccgtcgctcg tgatttgttt gcaaaaagaa
     1321 caaaactgaa aaaacccaga cacgctcgac ttcctgtctt cctattgatt gcagcttcca
     1381 atttcgtcac acaacaaggt cctagcgacg gctcacaggt tttgtaacaa gcaatcgaag
     1441 gttctggaat ggcgggaaag ggtttagtac cacatgctat gatgcccact gtgatctcca
     1501 gagcaaagtt cgttcgatcg tactgttact ctctctcttt caaacagaat tgtccgaatc
     1561 gtgtgacaac aacagcctgt tctcacacac tcttttcttc taaccaaggg ggtggtttag
     1621 tttagtagaa cctcgtgaaa cttacattta catatatata aacttgcata aattggtcaa
     1681 tgcaagaaat acatatttgg tcttttctaa ttcgtagttt ttcaagttct tagatgcttt
     1741 ctttttctct tttttacaga tcatcaagga agtaattatc tactttttac aacaaattaa
     1801 ttaaaatgac actctttccc tacacgacgc tcttccgatc tggcgcgccg atcggaagag
     1861 cacacgtctg aactccagtc acgacgcaca aacacgacga cgtgagcgtc gcgctgagaa
     1921 acaagctcaa tggaaagctg caaacgaagg tcgtggatca ctgctaacgt gtggagatgt
     1981 cgaagagaat ccaggtcctg gtaggagggc ttttgtagaa agaaatacga acgaaacgaa
     2041 aatcagcgtt gccatcgctt tggacaaagc tcccttacct gaagagtcga attttattga
     2101 tgaacttata acttccaagc atacaaacca aaagggagaa caagtaatcc aagtagacac
     2161 gggaattgga ttcttggatc acatgtatca tgcactggct aaacatgcag gctggagctt
     2221 acgactttac tcaagaggtg atttaatcat cgatgatcat cacactgcag aagatactgc
     2281 tattgcactt ggtattgcat tcaagcaggc tatgagtaac tttgccggcg ttaaaagatt
     2341 tggacatgct tattgtccac ttgacgaagc tctttctaga agcgtagttg acttgtcggg
     2401 acggccctat gctgttatcg atttgggatt aaagcgtgaa aaggttgggg aattgtcctg
     2461 tgaaatgatc cctcacttac tatattcctt ttcggtagca gctggaatta ctttgcatgt
     2521 tacctgctta tatggtagta atgaccatca tcgtgctgaa agcgctttta aatctctggc
     2581 tgttgccatg cgcgcggcta ctagtcttac tggaagttct gaagtcccaa gcacgaaggg
     2641 agtgttgtaa agagtactga caataaaaag attcttgttt tcaagaactt gtcatttgta
     2701 tagttttttt atattgtagt tgttctattt taatcaaatg ttagcgtgat ttatattttt
     2761 tttcgcctcg acatcatctg cccagatgcg aagttaagtg cgcagaaagt aatatcatgc
     2821 gtcaatcgta tgtgaatgct ggtcgctata ctgctgtcga ttcgatacta acgccgccat
     2881 ccagtttaaa cgagctcgaa ttcatcgatg atatcagatc cactagtggc ctatgcggcc
     2941 gcggatctgc cggtctccct atagtgagtc gtattaattt cgataagcca ggttaacctg
     3001 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     3061 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     3121 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     3181 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     3241 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     3301 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     3361 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     3421 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     3481 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     3541 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     3601 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     3661 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     3721 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     3781 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     3841 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     3901 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     3961 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     4021 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     4081 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     4141 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     4201 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     4261 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     4321 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     4381 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     4441 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     4501 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     4561 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     4621 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     4681 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     4741 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     4801 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     4861 ctt
//
