LOCUS       Exported                6148 bp ds-DNA     circular SYN 03-MAR-2022
DEFINITION  E. coli expression of soluble MHC protein.
ACCESSION   .
VERSION     .
KEYWORDS    pTCF6 HLA-A*24:02
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6148)
  AUTHORS   Altman lab
  TITLE     Altman lab bacterial expression plasmids
  JOURNAL   (unpublished)
REFERENCE   2  (bases 1 to 6148)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 3, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6148
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     21..40
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    40..64
                     /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)."
     RBS             79..101
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1033..1050
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1103..1121)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1117..1164
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1201..1656
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1288..1307)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1498..1519
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1748..2563)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2471..2490
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2555..2574
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2685..3273
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3174..3193
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3427..3444
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3459..3598
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3524..3543)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3684..3706
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3700..3891)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4700..5782)
                     /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     5744..5763
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5783..5860)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6066..6085
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
       61 ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat ggctagcggt
      121 tctcattcta tgagatattt ctccacatcc gtgtcccggc ccggccgcgg ggagccccgc
      181 ttcatcgccg tgggctacgt ggacgacacg cagttcgtgc ggttcgacag cgacgccgcg
      241 agccagagga tggagccgcg ggcgccgtgg atagagcagg aggggccgga gtattgggac
      301 gaggagacag ggaaagtgaa ggcccactca cagactgacc gagagaacct gcggatcgcg
      361 ctccgctact acaaccagag cgaggccggt tctcacaccc tccagatgat gtttggctgc
      421 gacgtggggt cggacgggcg cttcctccgc gggtaccacc agtacgccta cgacggcaag
      481 gattacatcg ccctgaaaga ggacctgcgc tcttggaccg cggcggacat ggcggctcag
      541 atcaccaagc gcaagtggga ggcggcccat gtggcggagc agcagagagc ctacctggag
      601 ggcacgtgcg tggacgggct ccgcagatac ctggagaacg ggaaggagac gctgcagcgc
      661 acggaccccc ccaagacaca tatgacccac caccccatct ctgaccatga ggccactctg
      721 agatgctggg ccctgggctt ctaccctgcg gagatcacac tgacctggca gcgggatggg
      781 gaggaccaga cccaggacac ggagcttgtg gagaccaggc ctgcagggga tggaaccttc
      841 cagaagtggg cagctgtggt ggtaccttct ggagaggagc agagatacac ctgccatgtg
      901 cagcatgagg gtctgcccaa gcccctcacc ctgagatggg agccatcttc ccagcccgga
      961 tccctgcatc atattctgga tgcacagaaa atggtgtgga atcatcgtta aaagcttgcg
     1021 gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
     1081 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     1141 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     1201 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     1261 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     1321 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     1381 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     1441 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     1501 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     1561 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     1621 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     1681 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
     1741 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     1801 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     1861 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     1921 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     1981 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
     2041 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     2101 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
     2161 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     2221 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     2281 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     2341 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     2401 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     2461 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     2521 atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
     2581 tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg
     2641 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     2701 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     2761 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     2821 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     2881 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     2941 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3001 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3061 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3121 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3181 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3241 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3301 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     3361 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     3421 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     3481 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     3541 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     3601 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     3661 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     3721 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     3781 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     3841 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     3901 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     3961 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     4021 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     4081 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     4141 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     4201 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     4261 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     4321 ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat
     4381 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     4441 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     4501 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     4561 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     4621 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     4681 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4741 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     4801 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     4861 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     4921 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     4981 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     5041 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     5101 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     5161 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     5221 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     5281 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     5341 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     5401 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     5461 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     5521 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     5581 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     5641 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     5701 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     5761 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     5821 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     5881 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     5941 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     6001 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     6061 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     6121 gccacgatgc gtccggcgta gaggatcg
//
