LOCUS       sequence-328169-        6572 bp    DNA     circular SYN 14-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6572)
  AUTHORS   Garcia-Blanco MA, Jamison SF, Sharp PA
  TITLE     Identification and purification of a 62,000-dalton protein that 
            binds specifically to the polypyrimidine tract of introns.
  JOURNAL   Genes Dev. 1989 Dec . 3(12A):1874-86.
  PUBMED    2533575
REFERENCE   2  (bases 1 to 6572)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Genes Dev. 
            1989 Dec . 3(12A):1874-86."
COMMENT     Sequence Label: pGEX-2TK-PTB
FEATURES             Location/Qualifiers
     source          1..6572
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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             76..729
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     687..709
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             736..753
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(2440..2462)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2607..2625)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2693..2797
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2798..2866,2867..3658)
                     /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(3016..3035)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3829..4417
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4318..4337
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4571..4588
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4661..4738
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4739..5821
                     /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(4758..4777)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5870..5875,5876..5893,5894..5900)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5908..5924
                     /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     5932..5948
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5932..5948
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5944..6471
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5964..5981)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5964..5980)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5973..5995)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      121 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      181 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      241 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      301 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      361 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      421 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      481 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      541 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      601 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      661 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      721 cctccaaaat cggatctggt tccgcgtgga tctcgtcgtg catctgttgg atccccggga
      781 attcatatgg acggcattgt cccagatata gccgttggta caaagcgggg atctgacgag
      841 cttttctcta cttgtgtcac taacggaccg ttaatcatga gcagcaactc ggcttctgca
      901 gcaaacggaa atgacagcaa gaagttcaaa ggtgacagcc gaagtgcagg cgtcccctct
      961 agagtgatcc tcatccggaa gctccccatc gacgtcacgg agggggaagt catctccctg
     1021 gggctgccct ttgggaaggt caccaacctc ctgatgctga aggggaaaaa ccaggccttc
     1081 atcgagatga acacggagga ggctgccaac accatggtga actactacac ctcggtgacc
     1141 cctgtgctgc gcggccagcc catctacatc cagttctcca accacaagga gctgaagacc
     1201 gacagctctc ccaaccaggc gcgggcccag gcggccctgc aggcggtgaa ctcggtccag
     1261 tcggggaacc tggccttggc tgcctcggcg gcggccgtgg acgcagggat ggcgatggcc
     1321 gggcagagcc ccgtgctcag gatcatcgtg gagaacctct tctaccctgt gaccctggat
     1381 gtgctgcacc agattttctc caagttcggc acagtgttga agatcatcac cttcaccaag
     1441 aacaaccagt tccaggccct gctgcagtat gcggaccccg tgagcgccca gcacgccaag
     1501 ctgtcgctgg acgggcagaa catctacaac gcctgctgca cgctgcgcat cgacttttcc
     1561 aagctcacca gcctcaacgt caagtacaac aatgacaaga gccgtgacta cacacgccca
     1621 gacctgcctt ccggggacag ccagccctcg ctggaccaga ccatggccgc ggccttcggc
     1681 ctttccgttc cgaacgtcca cggcgccctg gcccccctgg ccatcccctc ggcggcggcg
     1741 gcagctgcgg cggcaggtcg gatcgccatc ccgggcctgg cgggggcagg aaattctgta
     1801 ttgctggtca gcaacctcaa cccagagaga gtcacacccc aaagcctctt tattcttttc
     1861 ggcgtctacg gtgacgtgca gcgcgtgaag atcctgttca ataagaagga gaacgcccta
     1921 gtgcagatgg cggacggcaa ccaggcccag ctggccatga gccacctgaa cgggcacaag
     1981 ctgcacggga agcccatccg catcacgctc tcgaagcacc agaacgtgca gctgccccgc
     2041 gagggccagg aggaccaggg cctgaccaag gactacggca actcacccct gcaccgcttc
     2101 aagaagccgg gctccaagaa cttccagaac atattcccgc cctcggccac gctgcacctc
     2161 tccaacatcc cgccctcagt ctccgaggag gatctcaagg tcctgttttc cagcaatggg
     2221 ggcgtcgtca aaggattcaa gttcttccag aaggaccgca agatggcact gatccagatg
     2281 ggctccgtgg aggaggcggt ccaggccctc attgacctgc acaaccacga cctcggggag
     2341 aaccaccacc tgcgggtctc cttctccaag tccaccatct agaattcatc gtgactgact
     2401 gacgatctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     2461 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     2521 gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg
     2581 agtgtataat tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     2641 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     2701 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     2761 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     2821 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     2881 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     2941 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     3001 cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca
     3061 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     3121 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     3181 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     3241 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     3301 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt
     3361 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     3421 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     3481 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     3541 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     3601 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     3661 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     3721 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     3781 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3841 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3901 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3961 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4021 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4081 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4141 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4201 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4261 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4321 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4381 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4441 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4501 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     4561 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     4621 ccttacgcat ctgtgcggta tttcacaccg cataaattcc gacaccatcg aatggcgcaa
     4681 aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt
     4741 gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc
     4801 ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc
     4861 gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc
     4921 gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc
     4981 ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg
     5041 aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg
     5101 gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac
     5161 taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt
     5221 ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca
     5281 aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg
     5341 gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag
     5401 tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc
     5461 gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg
     5521 gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg
     5581 ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt
     5641 ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt
     5701 ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc
     5761 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     5821 agcgcaacgc aattaatgta agttagctca ctcattaggc accccaggct ttacacttta
     5881 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     5941 gctatgacca tgattacgga ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     6001 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     6061 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     6121 cgctttgcct ggtttccggc accagaagcg gtgccggaaa gctggctgga gtgcgatctt
     6181 cctgaggccg atactgtcgt cgtcccctca aactggcaga tgcacggtta cgatgcgccc
     6241 atctacacca acgtgaccta tcccattacg gtcaatccgc cgtttgttcc cacggagaat
     6301 ccgacgggtt gttactcgct cacatttaat gttgatgaaa gctggctaca ggaaggccag
     6361 acgcgaatta tttttgatgg cgttggaatt agcttatcga ctgcacggtg caccaatgct
     6421 tctggcgtca ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta aatcactgca
     6481 taattcgtgt cgctcaaggc gcactcccgt tctggataat gttttttgcg ccgacatcat
     6541 aacggttctg gcaaatattc tgaaatgagc tg
//
