LOCUS       sequence-352198-        7162 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses H6-MBP-LIF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7162)
  TITLE     Bacterial expression of Leukemia inhibitory factor as a maltose 
            binding protein fusion protein
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7162)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pMBP-hLIF
FEATURES             Location/Qualifiers
     source          1..7162
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        3..80
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             81..1163
                     /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(100..119)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1406..1411,1412..1427,1428..1434)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1442..1458
                     /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     1447..1469
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1546..1563
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1573..2670
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      2527..2536
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2644..2667
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2677..2697
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3253..3270)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3253..3269)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3262..3284)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3448..3465)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3618..3704
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3796..3823
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3842..3933
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3934..4002,4003..4794)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4152..4171)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(4836..5349)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5031..5052)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5243..5262
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      5460..6048
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5949..5968
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6202..6219
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6234..6376
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6302..6321)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6462..6484
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6478..6669)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
       61 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
      121 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
      181 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
      241 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
      301 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
      361 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
      421 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
      481 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
      541 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
      601 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
      661 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
      721 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
      781 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
      841 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcagta gtgggatacg
      901 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
      961 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     1021 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     1081 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     1141 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag
     1201 gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg
     1261 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg
     1321 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
     1381 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga
     1441 attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga
     1501 gcacttcacc aacaaggacc atagcatatg gggtctgata aaattcatca tcaccatcac
     1561 catggtagct ctaaaatcga agaaggtaaa ctggtaatct ggattaacgg cgataaaggc
     1621 tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc
     1681 gttgagcatc cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc
     1741 cctgacatta tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg
     1801 gctgaaatca ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc
     1861 gtacgttata acggcaaact gattgcttac cccatcgctg ttgaagcgtt atcgctgatt
     1921 tataacaaag atcttctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat
     1981 aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc
     2041 acctggccgc tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac
     2101 gacattaaag acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt
     2161 gacctgatta aaaacaaaca catgaatgca gacaccgatt actccatcgc agaagctgcc
     2221 tttaataaag gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac
     2281 accagcaaag tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa
     2341 ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca
     2401 aaagagttcc tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac
     2461 aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag agttggcgaa agatccacgt
     2521 attgccgcca ccatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg
     2581 tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact
     2641 gtcgatgaag ccctgaaaga cgcgcagact ggatccgaga atctgtactt ccagggttcc
     2701 cccctcccca tcacccctgt caacgccacc tgtgccattc gtcacccatg tcacaacaac
     2761 ctcatgaacc agatccgcag ccaactggca cagctcaatg gcagtgccaa tgccctcttt
     2821 attctctatt acacagccca gggggagccg ttccccaaca acctggacaa gctttgtggc
     2881 cccaacgtga cggacttccc gcccttccac gccaacggca cggagaaggc caagctggtg
     2941 gagctgtacc gcattgtcgt gtaccttggc acctccctgg gcaacatcac ccgtgaccag
     3001 aagatcctca accccagtgc cctcagcctc cacagcaagc tcaacgccac cgccgacatc
     3061 ctgcgtggcc tccttagcaa cgtgctgtgc cgcctgtgca gcaagtacca cgtgggccat
     3121 gtggacgtga cctacggccc tgacacctcg ggtaaggatg tcttccagaa gaagaagctg
     3181 ggctgtcaac tcctggggaa gtataagcag atcatcgccg tgttggccca ggccttctaa
     3241 tgaaagcttg gcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     3301 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     3361 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc agcttggctg
     3421 ttttggcgga tgagataaga ttttcagcct gatacagatt aaatcagaac gcagaagcgg
     3481 tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg accccatgcc
     3541 gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc atgcgagagt
     3601 agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt
     3661 ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt
     3721 tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca
     3781 ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt ctacaaactc
     3841 ttttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     3901 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     3961 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     4021 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     4081 aacagcggta agatccttga gagttttcgc cccgaagaac gtttcccaat gatgagcact
     4141 tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc
     4201 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     4261 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     4321 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     4381 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     4441 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     4501 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     4561 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     4621 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     4681 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     4741 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     4801 gaccaagttt actcatatat actttagatt gatttacccc ggttgataat cagaaaagcc
     4861 ccaaaaacag gaagattgta taagcaaata tttaaattgt aaacgttaat attttgttaa
     4921 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     4981 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     5041 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     5101 agggcgatgg cccactacgt gaaccatcac ccaaatcaag ttttttgggg tcgaggtgcc
     5161 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     5221 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     5281 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     5341 agggcgcgta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     5401 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     5461 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     5521 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     5581 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     5641 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     5701 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     5761 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     5821 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     5881 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     5941 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6001 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6061 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     6121 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     6181 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     6241 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg tgcactctca
     6301 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     6361 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     6421 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     6481 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     6541 gtcgtgcagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     6601 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     6661 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     6721 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     6781 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     6841 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     6901 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     6961 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     7021 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     7081 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggcca
     7141 ggacccaacg ctgcccgaaa tt
//
