LOCUS       sequence-421993-        5739 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector backbone for random polymerase chain reaction screen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5739)
  TITLE     Pospiech Lab Plasmids 2023
REFERENCE   2  (bases 1 to 5739)
  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"
COMMENT     Sequence Label: pEGGsH6_MCS_SHORT_outFrame
FEATURES             Location/Qualifiers
     source          1..5739
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(184..189,190..205,206..212)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    220..236
                     /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)."
     CDS             259..912
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     870..892
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             919..936
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             975..995
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(1002..1004,1005..1007,1008..1520)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGVVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIE"
     primer_bind     complement(1308..1327)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1655..1676
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1701..1721
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     primer_bind     complement(1790..1812)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1957..1975)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2043..2147
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2148..2216,2217..3008)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2366..2385)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3179..3767
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3668..3687
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3921..3938
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4011..4088
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4089..5171
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4108..4127)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5220..5225,5226..5243,5244..5250)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5258..5274
                     /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)."
     primer_bind     5282..5298
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5282..5298
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5294..5739,1..82)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5314..5331)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5314..5330)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5323..5345)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 tagcttatcg actgcacggt gcaccaatgc ttctggcgtc aggcagccat cggaagctgt
       61 ggtatggctg tgcaggtcgt aaatcactgc ataattcgtg tcgctcaagg cgcactcccg
      121 ttctggataa tgttttttgc gccgacatca taacggttct ggcaaatatt ctgaaatgag
      181 ctgttgacaa ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc
      241 acacaggaaa cagtattcat gtcccctata ctaggttatt ggaaaattaa gggccttgtg
      301 caacccactc gacttctttt ggaatatctt gaagaaaaat atgaagagca tttgtatgag
      361 cgcgatgaag gtgataaatg gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat
      421 cttccttatt atattgatgg tgatgttaaa ttaacacagt ctatggccat catacgttat
      481 atagctgaca agcacaacat gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg
      541 cttgaaggag cggttttgga tattagatac ggtgtttcga gaattgcata tagtaaagac
      601 tttgaaactc tcaaagttga ttttcttagc aagctacctg aaatgctgaa aatgttcgaa
      661 gatcgtttat gtcataaaac atatttaaat ggtgatcatg taacccatcc tgacttcatg
      721 ttgtatgacg ctcttgatgt tgttttatac atggacccaa tgtgcctgga tgcgttccca
      781 aaattagttt gttttaaaaa acgtattgaa gctatcccac aaattgataa gtacttgaaa
      841 tccagcaagt atatagcatg gcctttgcag ggctggcaag ccacgtttgg tggtggcgac
      901 catcctccaa aatcggatct ggttccgcgt ggatccgtcg acatatgcgc ggccgcagat
      961 cttggctagc cggcgaaaac ctgtatttcc aatcgcgacc catggtgagc aagggcgagg
     1021 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgtcgta aacggccaca
     1081 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     1141 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     1201 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     1261 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     1321 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     1381 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     1441 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     1501 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     1561 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     1621 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     1681 ccgccgggat cacccccggg caccatcatc accaccatca tagttaatta agaattcatc
     1741 gtgactgact gacgatctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     1801 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     1861 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta
     1921 gcgatagcgg agtgtataat tcttgaagac gaaagggcct cgtgatacgc ctatttttat
     1981 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     2041 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     2101 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     2161 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     2221 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     2281 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     2341 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg
     2401 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     2461 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     2521 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     2581 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     2641 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
     2701 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     2761 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     2821 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     2881 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     2941 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     3001 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     3061 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     3121 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3181 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     3241 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     3301 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     3361 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3421 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3481 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     3541 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     3601 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     3661 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     3721 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     3781 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     3841 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     3901 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     3961 ggtattttct ccttacgcat ctgtgcggta tttcacaccg cataaattcc gacaccatcg
     4021 aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg
     4081 tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc
     4141 agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag
     4201 tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg
     4261 gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc
     4321 aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga
     4381 tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac
     4441 gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag
     4501 ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca
     4561 gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg
     4621 gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc
     4681 tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag
     4741 gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg
     4801 ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta
     4861 ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag
     4921 acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc
     4981 aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc
     5041 tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct
     5101 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     5161 gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc accccaggct
     5221 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     5281 acaggaaaca gctatgacca tgattacgga ttcactggcc gtcgttttac aacgtcgtga
     5341 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     5401 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     5461 tggcgaatgg cgctttgcct ggtttccggc accagaagcg gtgccggaaa gctggctgga
     5521 gtgcgatctt cctgaggccg atactgtcgt cgtcccctca aactggcaga tgcacggtta
     5581 cgatgcgccc atctacacca acgtgaccta tcccattacg gtcaatccgc cgtttgttcc
     5641 cacggagaat ccgacgggtt gttactcgct cacatttaat gttgatgaaa gctggctaca
     5701 ggaaggccag acgcgaatta tttttgatgg cgttggaat
//
